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Sample records for quadruplexes sequence effects

  1. Conformation and Stability of Intramolecular Telomeric G-Quadruplexes: Sequence Effects in the Loops

    Science.gov (United States)

    Sattin, Giovanna; Artese, Anna; Nadai, Matteo; Costa, Giosuè; Parrotta, Lucia; Alcaro, Stefano; Palumbo, Manlio; Richter, Sara N.

    2013-01-01

    Telomeres are guanine-rich sequences that protect the ends of chromosomes. These regions can fold into G-quadruplex structures and their stabilization by G-quadruplex ligands has been employed as an anticancer strategy. Genetic analysis in human telomeres revealed extensive allelic variation restricted to loop bases, indicating that the variant telomeric sequences maintain the ability to fold into G-quadruplex. To assess the effect of mutations in loop bases on G-quadruplex folding and stability, we performed a comprehensive analysis of mutant telomeric sequences by spectroscopic techniques, molecular dynamics simulations and gel electrophoresis. We found that when the first position in the loop was mutated from T to C or A the resulting structure adopted a less stable antiparallel topology; when the second position was mutated to C or A, lower thermal stability and no evident conformational change were observed; in contrast, substitution of the third position from A to C induced a more stable and original hybrid conformation, while mutation to T did not significantly affect G-quadruplex topology and stability. Our results indicate that allelic variations generate G-quadruplex telomeric structures with variable conformation and stability. This aspect needs to be taken into account when designing new potential anticancer molecules. PMID:24367632

  2. Conformation and stability of intramolecular telomeric G-quadruplexes: sequence effects in the loops.

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    Giovanna Sattin

    Full Text Available Telomeres are guanine-rich sequences that protect the ends of chromosomes. These regions can fold into G-quadruplex structures and their stabilization by G-quadruplex ligands has been employed as an anticancer strategy. Genetic analysis in human telomeres revealed extensive allelic variation restricted to loop bases, indicating that the variant telomeric sequences maintain the ability to fold into G-quadruplex. To assess the effect of mutations in loop bases on G-quadruplex folding and stability, we performed a comprehensive analysis of mutant telomeric sequences by spectroscopic techniques, molecular dynamics simulations and gel electrophoresis. We found that when the first position in the loop was mutated from T to C or A the resulting structure adopted a less stable antiparallel topology; when the second position was mutated to C or A, lower thermal stability and no evident conformational change were observed; in contrast, substitution of the third position from A to C induced a more stable and original hybrid conformation, while mutation to T did not significantly affect G-quadruplex topology and stability. Our results indicate that allelic variations generate G-quadruplex telomeric structures with variable conformation and stability. This aspect needs to be taken into account when designing new potential anticancer molecules.

  3. A library screening approach identifies naturally occurring RNA sequences for a G-quadruplex binding ligand.

    Science.gov (United States)

    Mirihana Arachchilage, Gayan; Morris, Mark J; Basu, Soumitra

    2014-02-07

    An RNA G-quadruplex library was synthesised and screened against kanamycin A as the ligand. Naturally occurring G-quadruplex forming sequences that differentially bind to kanamycin A were identified and characterized. This provides a simple and effective strategy for identification of potential intracellular G-quadruplex targets for a ligand.

  4. Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression.

    Science.gov (United States)

    Li, Yue; Syed, Junetha; Suzuki, Yuki; Asamitsu, Sefan; Shioda, Norifumi; Wada, Takahito; Sugiyama, Hiroshi

    2016-05-17

    ATR-X (α-thalassemia/mental retardation X-linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α-globin gene cluster. The VNTR sequence, which contains the potential G-quadruplex-forming sequence CGC(GGGGCGGGG)n , is involved in the downregulation of α-globin expression. We investigated G-quadruplex and i-motif formation in single-stranded DNA and long double-stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G-quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G-quadruplex formation by the VNTR sequence downregulates the expression of α-globin genes and that ATRX might bind to and resolve the G-quadruplex.

  5. Quadruplexes of human telomere dG{sub 3}(TTAG{sub 3}){sub 3} sequences containing guanine abasic sites

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    Skolakova, Petra; Bednarova, Klara; Vorlickova, Michaela [Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-612 65 Brno (Czech Republic); Sagi, Janos, E-mail: jans@rimstonelab.com [Rimstone Laboratory, RLI, 29 Lancaster Way, Cheshire, CT 06410 (United States)

    2010-08-20

    Research highlights: {yields} Loss of a guanine base does not hinder the formation of G-quadruplex of human telomere sequence. {yields} Each depurination strongly destabilizes the quadruplex of dG{sub 3}(TTAG{sub 3}){sub 3} in NaCl and KCl. {yields} Conformational change of the abasic analogs of dG{sub 3}(TTAG{sub 3}){sub 3} is inhibited in KCl. {yields} The effects abasic sites may affect telomere-end structures in vivo. -- Abstract: This study was performed to evaluate how the loss of a guanine base affects the structure and stability of the three-tetrad G-quadruplex of 5'-dG{sub 3}(TTAG{sub 3}){sub 3}, the basic quadruplex-forming unit of the human telomere DNA. None of the 12 possible abasic sites hindered the formation of quadruplexes, but all reduced the thermodynamic stability of the parent quadruplex in both NaCl and KCl. The base loss did not change the Na{sup +}-stabilized intramolecular antiparallel architecture, based on CD spectra, but held up the conformational change induced in dG{sub 3}(TTAG{sub 3}){sub 3} in physiological concentration of KCl. The reduced stability and the inhibited conformational transitions observed here in vitro for the first time may predict that unrepaired abasic sites in G-quadruplexes could lead to changes in the chromosome's terminal protection in vivo.

  6. Sequence, Stability, Structure of G-Quadruplexes and Their Drug Interactions

    Science.gov (United States)

    Chen, Yuwei; Yang, Danzhou

    2012-01-01

    Although DNA is most widely known to store and pass along genetic information, the discovery of G-quadruplex structures has illuminated a new role of DNA in biology. DNA G-quadruplexes are four-stranded globular nucleic acid secondary structures formed in specific G-rich sequences with biological significance, such as human telomeres and oncogene promoters. This review focuses on the unimolecular DNA G-quadruplexes, which can readily form in solution under physiological conditions and are considered to be most biologically relevant. Available structural data show a great conformational diversity of unimolecular G-quadruplexes, amenable to small molecule drug targeting. The relationship of sequence, structure, and stability of unimolecular DNA G-quadruplexes, as well as the recent progress on interactions with small molecule compounds and insights into rational design of G-quadruplex-interactive molecules, will be discussed. PMID:22956454

  7. Unfolding thermodynamics of intramolecular G-quadruplexes: base sequence contributions of the loops.

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    Olsen, Chris M; Lee, Hui-Ting; Marky, Luis A

    2009-03-05

    G-quadruplexes are a highly studied DNA motif with a potential role in a variety of cellular processes and more recently are considered novel targets for drug therapy in aging and anticancer research. In this work, we have investigated the thermodynamic contributions of the loops on the stable formation of G-quadruplexes. Specifically, we use a combination of UV, circular dichroism (CD) and fluorescence spectroscopies, and differential scanning calorimetry (DSC) to determine thermodynamic profiles, including the differential binding of ions and water, for the unfolding of the thrombin aptamer: d(GGT2GGTGTGGT2GG) that is referred to as G2. The sequences in italics, TGT and T2, are known to form loops. Other sequences examined contained base substitutions in the TGT loop (TAT, TCT, TTT, TAPT, and UUU), in the T2 loops (T4, U2), or in both loops (UGU and U2, UUU and U2). The CD spectra of all molecules show a positive band centered at 292 nm, which corresponds to the "chair" conformation. The UV and DSC melting curves of each G-quadruplex show monophasic transitions with transition temperatures (T(M)s) that remained constant with increasing strand concentration, confirming their intramolecular formation. These G-quadruplexes unfold with T(M)s in the range from 43.2 to 56.5 degrees C and endothermic enthalpies from 22.9 to 37.2 kcal/mol. Subtracting the contribution of a G-quartet stack from each experimental profile indicated that the presence of the loops stabilize each G-quadruplex by favorable enthalpy contributions, larger differential binding of K+ ions (0.1-0.6 mol K+/ mol), and a variable uptake/release of water molecules (-6 to 8 mol H2O/mol). The thermodynamic contributions for these specific base substitutions are discussed in terms of loop stacking (base-base stacking within the loops) and their hydration effects.

  8. Effect of G-quadruplex polymorphism on the recognition of telomeric DNA by a metal complex.

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    Caterina Musetti

    Full Text Available The physiological role(s played by G-quadruplexes renders these 'non-canonical' DNA secondary structures interesting new targets for therapeutic intervention. In particular, the search for ligands for selective recognition and stabilization of G-quadruplex arrangements has led to a number of novel targeted agents. An interesting approach is represented by the use of metal-complexes, their binding to DNA being modulated by ligand and metal ion nature, and by complex stoichiometry. In this work we characterized thermodynamically and stereochemically the interactions of a Ni(II bis-phenanthroline derivative with telomeric G-quadruplex sequences using calorimetric, chiroptical and NMR techniques. We employed three strictly related sequences based on the human telomeric repeat, namely Tel22, Tel26 and wtTel26, which assume distinct conformations in potassium containing solutions. We were able to monitor specific enthalpy/entropy changes according to the structural features of the target telomeric sequence and to dissect the binding process into distinct events. Interestingly, temperature effects turned out to be prominent both in terms of binding stoichiometry and ΔH/ΔS contributions, while the final G-quadruplex-metal complex architecture tended to merge for the examined sequences. These results underline the critical choice of experimental conditions and DNA sequence for practical use of thermodynamic data in the rational development of effective G-quadruplex binders.

  9. Separation of Quadruplex Polymorphism in DNA Sequences by Reversed-Phase Chromatography

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    Miller, M. Clarke; Ohrenberg, Carl J.; Kuttan, Ashani; Trent, John O.

    2015-01-01

    This unit describes a method for the separation of a mixture of quadruplex conformations formed from the same parent sequence via reversed-phase chromatography (RPC). Polymorphism is inherent to quadruplex formation and even relatively simple quadruplex-forming sequences can fold into a cornucopia of possible conformations and topologies. Isolation of a specific conformation for study can be problematic. This is especially true for conformations of the human telomere sequence d(GGG(TTAGGG)3), High Performance Liquid Chromatography (HPLC), especially reversed-phase chromatography, has been a mainstay of nucleic acids research and purification for many decades. We have successfully applied this method to the problem of separating individual quadruplex species in the ensemble from the same parent sequence. PMID:26344226

  10. Not All G-Quadruplexes are Created Equally: An Investigation of the Structural Polymorphism of the c-Myc G-Quadruplex-Forming Sequence and its Interaction with the Porphyrin meso-Tetra(N-methyl-4-pyridyl)porphine

    Science.gov (United States)

    Le, Huy T.; Miller, M. Clarke; Buscaglia, Robert; Dean, William L.; Holt, Patrick A.; Chaires, Jonathan B.; Trent, John O.

    2012-01-01

    G-quadruplexes, DNA tertiary structures highly localized to functionally important sites within the human genome, have emerged as important new drug targets. The putative G-quadruplex-forming sequence (Pu27) in the NHE-III1 promoter region of the c-Myc gene is of particular interest as stabilization of this G-quadruplex with TMPyP4 has been shown to repress c-Myc transcription. In this study, we examine the Pu27 G-quadruplex-forming sequence and its interaction with TMPyP4. We report that the Pu27 sequence exists as a heterogeneous mixture of monomeric and higher-order G-quadruplex species in vitro and that this mixture can be partially resolved by size exclusion chromatography (SEC) separation. Within this ensemble of configurations, the equilibrium can be altered by modifying the buffer composition, annealing procedure, and dialysis protocol thereby affecting the distribution of G-quadruplex species formed. TMPyP4 was found to bind preferentially to higher-order G-quadruplex species suggesting the possibility of stabilization of the junctions of the c-Myc G-quadruplex multimers by porphyrin end-stacking. We also examined four modified c-Myc sequences that have been previously reported and found a narrower distribution of quadruplex configurations compared to the parent Pu27 sequence. We could not definitively conclude whether these G-quadruplex structures were selected from the original ensemble or if they are new G-quadruplex structures. Since these sequences differ considerably from the wild-type promoter sequence, it is unclear whether their structures have any actual biological relevance. Additional studies are needed to examine how the polymorphic nature of G-quadruplexes affects the interpretation of in vitro data for c-Myc and other G-quadruplexes. The findings reported here demonstrate that experimental conditions contribute significantly to G-quadruplex formation and should be carefully considered, controlled, and reported in detail. PMID:23108607

  11. Selection of G-quadruplex folding topology with LNA-modified human telomeric sequences in K+ solution

    DEFF Research Database (Denmark)

    Pradhan, Devranjan; Hansen, Lykke H; Vester, Birte

    2011-01-01

    this problem by examining the impact of LNA (locked nucleic acid) modifications on the folding topology of the dimeric model system of the human telomere sequence. In solution, this DNA G-quadruplex forms a mixture of G-quadruplexes with antiparallel and parallel topologies. Using CD and NMR spectroscopies, we...

  12. Formation of highly ordered multimers in G-quadruplexes.

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    Tóthová, Petra; Krafčíková, Petra; Víglaský, Viktor

    2014-11-18

    G-Rich DNA and RNA have a higher propensity to form G-quadruplex structures, but the presence of G-runs alone is not sufficient to prove that such sequences can form stable G-quadruplexes. While G-rich sequences are essential for G-quadruplex formation, not all G-rich sequences have the propensity to form G-quadruplex structures. In addition, monovalent metal ions, dehydrating agents, and loop sequences connecting the G-runs also play important roles in the topology of G-quadruplex folding. To date, no quantitative analysis of the CD spectra of G-quadruplexes in confrontation with the electrophoretic results has been performed. Therefore, in this study, we use information gained through the analysis of a series of well-known G-quadruplex-forming sequences to evaluate other less-studied sets of aptameric sequences. A simple and cost-effective methodology that can verify the formation of G-quadruplex motifs from oligomeric DNA sequences and a technique to determine the molecularity of these structures are also described. This methodology could be of great use in the prediction of G-quadruplex assembly, and the basic principles of our techniques can be extrapolated for any G-rich DNA sequences. This study also presents a model that can predict the multimerization of G-quadruplexes; the predictions offered by this model are shown to match the results obtained using circular dichroism.

  13. Ru-indoloquinoline complex as a selective and effective human telomeric G-quadruplex binder

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    Yu, Hui-juan; Zhao, Ying; Mo, Wei-jie; Hao, Zhi-feng; Yu, Lin

    2014-11-01

    Indoloquinoline and its derivatives have been reported to be a kind of efficient G-quadruplex binder and have been found to interact preferentially to intramolecular G-quadruplex and inhibit telomerase activity in human K562 cells and SW620 cells. In contrast to indoloquinoline derivatives, much less is known about the metal complex based on indoloquinoline or its derivative. In this report, we studied the interaction of ruthenium complex [Ru(bpy)2(itatp)]2+ containing indoloquinoline moiety with human telomeric G-quadruplex DNA (Telo22) and c-myc G-quadruplex DNA (Pu27) by UV-visible (UV-Vis), fluorescence spectroscopy, fluorescent intercalator displacement (FID), thermal denaturation studies and CD spectroscopy. The results suggest that [Ru(bpy)2(itatp)]2+ displays a strong π-π stacking interaction with human telomeric G-quadruplex with a high binding constant (∼107 M-1), but just exhibits moderate binding affinity to c-myc G-quadruplex, thus showing significant selectivity to human telomeric G-quadruplex. The CD titration results indicate that [Ru(bpy)2(itatp)]2+ could effectively convert Telo22 into antiparallel G-quadruplex conformation, while in the c-myc G-quadruplex case, instead of promoting Pu27 to fold into G-quadruplex, [Ru(bpy)2(itatp)]2+ destroys the parallel G-quadruplex structure of Pu27.

  14. Assessing the amount of quadruplex structures present within G₂-tract synthetic random-sequence DNA libraries.

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    McManus, Simon A; Li, Yingfu

    2013-01-01

    The process of in vitro selection has led to the discovery of many aptamers with potential to be developed into inhibitors and biosensors, but problems in isolating aptamers against certain targets with desired affinity and specificity still remain. One possible improvement is to use libraries enhanced for motifs repeatedly isolated in aptamer molecules. One such frequently observed motif is the two-tiered guanine quadruplex. In this study we investigated whether DNA libraries could be designed to contain a large fraction of molecules capable of folding into two-tiered guanine quadruplexes. Using comprehensive circular dichroism analysis, we found that DNA libraries could be designed to contain a large proportion of sequences that adopt guanine quadruplex structures. Analysis of individual sequences from a small library revealed a mixture of quadruplexes of different topologies providing the diversity desired for an in vitro selection. We also found that primer-binding sites are detrimental to quadruplex formation and devised a method for post-selection amplification of primer-less quadruplex libraries. With the development of guanine quadruplex enriched DNA libraries, it should be possible to improve the chances of isolating aptamers that utilize a quadruplex scaffold and enhance the success of in vitro selection experiments.

  15. Effects of metal ions and cosolutes on G-quadruplex topology.

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    Fujii, Taiga; Podbevšek, Peter; Plavec, Janez; Sugimoto, Naoki

    2017-01-01

    Topologies of G-quadruplexes depend on oligonucleotide sequences and on environmental factors, and the diversity of G-quadruplex topologies complicates investigation of functions of these nucleic acid structures. To investigate how metal ions and cosolutes regulate topologies of G-quadruplexes, we stabilized the antiparallel conformation by insertion of 2'-deoxyxanthosine and 8-oxo-2'-deoxyguanosine into selected positions of an oligonucleotide. Thermodynamic analyses of the oligonucleotide revealed that Na(+) stabilized the antiparallel G-quadruplex, whereas K(+) destabilized this topology. This result suggests that metal ions selectively stabilize G-quadruplex topologies with cavities between G-quartet planes of certain sizes. In the presence of KCl in 20wt% poly(ethylene glycol) with average molecular weight of 200, the antiparallel basket-type G-quadruplex conformation was not stabilized compared with the dilute condition. In the presence of NaCl, the cosolute did stabilize the G-quadruplex with respect to the dilute condition. The presented data show that metal ions and cosolutes regulate topologies of G-quadruplexes through mechanisms that depend on sizes of metal ion cavities and hydration states.

  16. Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution

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    Ambrus, Attila; Chen, Ding; Dai, Jixun; Bialis, Tiffanie; Jones, Roger A.; Yang, Danzhou

    2006-01-01

    Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug ...

  17. Effects of monovalent cations on folding kinetics of G-quadruplexes.

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    You, Jing; Li, Hui; Lu, Xi-Ming; Li, Wei; Wang, Peng-Ye; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-08-31

    G-quadruplexes are special structures existing at the ends of human telomeres, the folding kinetics of which are essential for their functions, such as in the maintenance of genome stability and the protection of chromosome ends. In the present study, we investigated the folding kinetics of G-quadruplex in different monovalent cation environments and determined the detailed kinetic parameters for Na(+)- and K(+)-induced G-quadruplex folding, and for its structural transition from the basket-type Na(+) form to the hybrid-type K(+) form. More interestingly, although Li(+) was often used in previous studies of G-quadruplex folding as a control ion supposed to have no effect, we have found that Li(+) can actually influence the folding kinetics of both Na(+)- and K(+)-induced G-quadruplexes significantly and in different ways, by changing the folding fraction of Na(+)-induced G-quadruplexes and greatly increasing the folding rates of K(+)-induced G-quadruplexes. The present study may shed new light on the roles of monovalent cations in G-quadruplex folding and should be useful for further studies of the underlying folding mechanism. © 2017 The Author(s).

  18. The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo.

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    Cyril Ribeyre

    2009-05-01

    Full Text Available In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4 secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Delta cells. Hence, we conclude that CEB1 instability in pif1Delta cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.

  19. Spectroscopic investigation of the interaction between G-quadruplex of KRAS promoter sequence and three isoquinoline alkaloids

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    Wen, Li-Na; Xie, Meng-Xia

    2017-01-01

    KRAS promoter can form G-quadruplex structure and regulate gene transcription. The drugs which can bind with G-quadruplex of KRAS promoter may be potential remedy for treatment of cancers associated with KRAS mutation. The interaction mechanism between the G-quadruplex of KRAS promoter and three isoquinoline alkaloids (jatrorrhizine, berberine and sanguinarine) has been investigated by UV-visible, fluorescence and circular dichroism spectroscopic methods. The results showed that the three alkaloids can form complexes with G-quadruplex KRAS promoter with the molecular ratio of 1:1, and the binding constants were (0.90 ± 0.16) × 106 L mol- 1, (0.93 ± 0.21) × 106 L mol- 1 and (1.16 ± 0.45) × 106 L mol- 1 for jatrorrhizine, berberine and sanguinarine. The absorption spectra, KI quenching and fluorescence anisotropy and polarization studies suggested jatrorrhizine and berberine interacted with G-quadruplex by not only end-stacking binding mode but also grooves or loops binding mode, while sanguinarine by end-stacking binding mode. Sanguinarine was more beneficial to maintain the stability and parallel conformation of KRAS promoter G-quadruplex. MTT assay was performed to evaluate antiproliferation effects of the three isoquinoline alkaloids on SW620 cells, and the antiproliferation effects of the three alkaloids were sanguinarine > berberine > jatrorrhizine. All the three alkaloids can bind with KRAS promoter G-quadruplex, and sanguinarine had the better binding property and antiproliferation effects on SW620 cells. The results obtained are meaningful to explore potential reagents targeting the parallel G-quadruplex structure of KRAS promoter for gene theraphy of colorectal carcinomas.

  20. Right-handed and left-handed G-quadruplexes have the same DNA sequence: distinct conformations induced by an organic small molecule and potassium.

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    Fu, Boshi; Huang, Jinguo; Chen, Yuqi; Wang, Yafen; Xue, Tianrui; Xu, GuoHua; Wang, Shaoru; Zhou, Xiang

    2016-08-21

    Herein, we report two distinct G-quadruplex conformations of the same G-rich oligonucleotide, regulated by a small molecule. This is the first report in which both right- and left-handed G-quadruplex conformations have been obtained from the same sequence. We discriminated these two distinct conformations and investigated their kinetics and thermodynamics.

  1. Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

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    Rehm, Charlotte; Wurmthaler, Lena A; Li, Yuanhao; Frickey, Tancred; Hartig, Jörg S

    2015-01-01

    In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1-5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria.

  2. The Truncated Human Telomeric Sequence forms a Hybrid-Type Intramolecular Mixed Parallel/antiparallel G-quadruplex Structure in K(+) Solution.

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    Liu, Yuxia; Cheng, Dengfeng; Ge, Min; Lin, Weizhen

    2016-07-01

    In 80-90% tumor cells, telomerase becomes active and stabilizes the length of telomeres. The formation and stabilization of G-quadruplexes formed from human telomeric sequences have been proved able to inhibit the activity of telomerase, thus human telomeric G-quadruplex structure has become a potential target for the development of cancer therapy. Hence, structure of G-quadruplex formed in K(+) solution has been an attractive hotspot for further studies. However, the exact structure of human telomeric G-quadruplex in K(+) is extremely controversial, this study provides information for the understanding of different G-quadruplexes. Here, we report that 22nt and 24nt human telomeric sequences form unimolecular hybrid-type mixed parallel/antiparallel G-quadruplex in K(+) solution elucidated utilizing Circular Dichroism, Differential Scanning Calorimetry, and gel electrophoresis. Moreover, individual configuration of these two sequences was speculated in this study. The detailed structure information of the G-quadruplex formed under physiologically relevant condition is necessary for structure-based rational drug design.

  3. Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

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    Charlotte Rehm

    Full Text Available In prokaryotes simple sequence repeats (SSRs with unit sizes of 1-5 nucleotides (nt are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4 structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc, Xanthomonas axonopodis pv. citri str. 306 (Xac, and Nostoc sp. strain PCC7120 (Ana. In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria.

  4. Structural and functional analysis of DNA sequences with potential for forming G-quadruplex

    OpenAIRE

    Luciana Souto Mofatto

    2013-01-01

    Resumo: Os G-quadruplexes são estruturas secundárias de DNA altamente organizadas, constituídas por sequências ricas em guaninas capazes de formar tétrades ligadas por pontes de hidrogênio. Essas sequências são capazes de modular a transcrição gênica e o splicing alternativo de éxons. Além disso, estudos também mostraram que os G-quadruplexes estão presentes na região promotora de oncogenes (como c-MYC) e nas regiões terminais dos telômeros, indicando que o G-quadruplex pode ser um possível a...

  5. In silico identification of novel ligands for G-quadruplex in the c-MYC promoter.

    Science.gov (United States)

    Kang, Hyun-Jin; Park, Hyun-Ju

    2015-04-01

    G-quadruplex DNA formed in NHEIII1 region of oncogene promoter inhibits transcription of the genes. In this study, virtual screening combining pharmacophore-based search and structure-based docking screening was conducted to discover ligands binding to G-quadruplex in promoter region of c-MYC. Several hit ligands showed the selective PCR-arresting effects for oligonucleotide containing c-MYC G-quadruplex forming sequence. Among them, three hits selectively inhibited cell proliferation and decreased c-MYC mRNA level in Ramos cells, where NHEIII1 is included in translocated c-MYC gene for overexpression. Promoter assay using two kinds of constructs with wild-type and mutant sequences showed that interaction of these ligands with the G-quadruplex resulted in turning-off of the reporter gene. In conclusion, combined virtual screening methods were successfully used for discovery of selective c-MYC promoter G-quadruplex binders with anticancer activity.

  6. Synthesis of New Benzimidazole and Benzothiazole Disulfide Metal Complexes as G-quadruplex Binding Ligands.

    Science.gov (United States)

    Saour, Kawkab; Lafta, Dunya

    2016-01-01

    Compounds that can bind and stabilize non-canonical DNA structures are named quadruplex and are of interest in anticancer drug design due to their selective inhibitions of telomerase and consequent effects on cell proliferation. In this study, we report novel Co/Cu [II] complex compounds as G-quadruplex DNA binding ligands. The results from the preliminary assay indicated that the introduction of a positively charged 6-membered tail to the aromatic terminal group of benzimidazole significantly enhanced the binding affinity with the quadruplex and exhibited anti-telomerase activity. These derivatives showed significant selectivities for the telomeric quadruplex over duplex nucleic acids. The stabilization of non-canonical forms estimated with the FRET DNA technology using different sequences, such as F21T, c-kit1 and c-kit2, in cancer cell lines were assessed. Three members of this family showed to be very selective in stabilizing one particular G-quadruplex.

  7. Recognition of human telomeric G-quadruplex DNA by berberine analogs: effect of substitution at the 9 and 13 positions of the isoquinoline moiety.

    Science.gov (United States)

    Bhowmik, Debipreeta; Fiorillo, Gaetano; Lombardi, Paolo; Kumar, G Suresh

    2015-12-01

    G-quadruplex forming sequences are widely distributed in human genome and serve as novel targets for regulating gene expression and chromosomal maintenance. They offer unique targets for anticancer drug development. Here, the interaction of berberine (BC) and two of its analogs bearing substitution at 9 and 13-position with human telomeric G-quadruplex DNA sequence has been investigated by biophysical techniques. Both the analogs exhibited several-fold higher binding affinity than berberine. The Scatchard binding isotherms revealed non-cooperative binding. 9-ω-amino hexyl ether analog (BC1) showed highest affinity (1.8 × 10(6) M(-1)) while the affinity of the 13-phenylpropyl analog (BC2) was 1.09 × 10(6) M(-1). Comparative fluorescence quenching and polarization anisotropy of the emission spectra gave evidence for a stronger stacking interaction of the analogs compared to berberine. The thiazole orange displacement assay has clearly established that the analogs were more effective in displacing the end stacked dye in comparison to berberine. However, the binding of the analogs did not induce any major structural perturbation in the G-quadruplex structure, but led to higher thermal stability. Energetics of the binding indicated that the association of the analogs was exothermic and predominantly entropy driven phenomenon. Increasing the temperature resulted in weaker binding; the enthalpic contribution increased and the entropic contribution decreased. A small negative heat capacity change with significant enthalpy-entropy compensation established the involvement of multiple weak noncovalent interactions in the binding process. The 9-ω-amino hexyl ether analog stabilized the G-quadruplex structure better than the 13-phenyl alkyl analog.

  8. c-Myc quadruplex-forming sequence Pu-27 induces extensive damage in both telomeric and nontelomeric regions of DNA.

    Science.gov (United States)

    Islam, Md Ashraful; Thomas, Shelia D; Murty, Vundavalli V; Sedoris, Kara J; Miller, Donald M

    2014-03-21

    Quadruplex-forming DNA sequences are present throughout the eukaryotic genome, including in telomeric DNA. We have shown that the c-Myc promoter quadruplex-forming sequence Pu-27 selectively kills transformed cells (Sedoris, K. C., Thomas, S. D., Clarkson, C. R., Muench, D., Islam, A., Singh, R., and Miller, D. M. (2012) Genomic c-Myc quadruplex DNA selectively kills leukemia. Mol. Cancer Ther. 11, 66-76). In this study, we show that Pu-27 induces profound DNA damage, resulting in striking chromosomal abnormalities in the form of chromatid or chromosomal breaks, radial formation, and telomeric DNA loss, which induces γ-H2AX in U937 cells. Pu-27 down-regulates telomeric shelterin proteins, DNA damage response mediators (RAD17 and RAD50), double-stranded break repair molecule 53BP1, G2 checkpoint regulators (CHK1 and CHK2), and anti-apoptosis gene survivin. Interestingly, there are no changes of DNA repair molecules H2AX, BRCA1, and the telomere maintenance gene, hTERT. ΔB-U937, where U937 cells stably transfected with deleted basic domain of TRF2 is partially sensitive to Pu-27 but exhibits no changes in expression of shelterin proteins. However, there is an up-regulation of CHK1, CHK2, H2AX, BRCA1, and survivin. Telomere dysfunction-induced foci assay revealed co-association of TRF1with γ-H2AX in ATM deficient cells, which are differentially sensitive to Pu-27 than ATM proficient cells. Alt (alternating lengthening of telomere) cells are relatively resistant to Pu-27, but there are no significant changes of telomerase activity in both Alt and non-Alt cells. Lastly, we show that this Pu-27-mediated sensitivity is p53-independent. The data therefore support two conclusions. First, Pu-27 induces DNA damage within both telomeric and nontelomeric regions of the genome. Second, Pu-27-mediated telomeric damage is due, at least in part, to compromise of the telomeric shelterin protein complex.

  9. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  10. Single-Molecule FRET Study of DNA G-Quadruplex

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The DNA G-quadruplex formed by the human telomeric sequence is a potential target for novel anticancer drugs. We have investigated an intramolecular DNA G-quadruplex using single-molecule fluorescence resonance energy transfer and shown that individual folded quadruplexes can be identified. The mean proximity ratio measured at the single-molecule level was consistent with ensemble measurement.

  11. Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method

    Science.gov (United States)

    Laguerre, Aurélien; Wong, Judy M. Y.; Monchaud, David

    2016-01-01

    Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities. PMID:27535322

  12. Nemorubicin and doxorubicin bind the G-quadruplex sequences of the human telomeres and of the c-MYC promoter element Pu22.

    Science.gov (United States)

    Scaglioni, Leonardo; Mondelli, Rosanna; Artali, Roberto; Sirtori, Federico Riccardi; Mazzini, Stefania

    2016-06-01

    Intra-molecular G-quadruplex structures are present in the guanine rich regions of human telomeres and were found to be prevalent in gene promoters. More recently, the targeting of c-MYC transcriptional control has been suggested, because the over expression of the c-MYC oncogene is one of the most common aberration found in a wide range of human tumors. The interaction of nemorubicin and doxorubicin with DNA G-quadruplex structures has been studied by NMR, ESI-MS and molecular modelling, in order to obtain further information about the complex and the multiple mechanisms of action of these drugs. Nemorubicin intercalates between A3 and G4 of d(TTAGGGT)4 and form cap-complex at the G6pT7 site. The presence of the adenine in this sequence is important for the stabilization of the complex, as was shown by the interaction with d(TTGGGTT)4 and d(TTTGGGT)4, which form only a 1:1 complex. The interaction of doxorubicin with d(TTAGGGT)4 is similar, but the complex appears less stable. Nemorubicin also binds with high efficiency the c-MYC G-quadruplex sequence Pu22, to form a very well defined complex. Two nemorubicin molecules bind to the 3'-end and to the 5'-end, forming an additional plane of stacking over each external G-tetrad. The wild type c-MYCPu22 sequence forms with nemorubicin the same complex. Nemorubicin and doxorubicin, not only intercalate into the duplex DNA, but also result in significant ligands for G-quadruplex DNA segments, stabilizing their structure; this may in part explain the multiple mechanisms of action of their antitumor activity. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quadruplex DNA.

    Science.gov (United States)

    Nanjunda, Rupesh; Owens, Eric A; Mickelson, Leah; Alyabyev, Sergey; Kilpatrick, Nancy; Wang, Siming; Henary, Maged; Wilson, W David

    2012-12-15

    Design and optimization of quadruplex-specific small molecules is developing into an attractive strategy for anti-cancer therapeutics with some promising candidates in clinical trials. A number of therapeutically favorable features of cyanine molecules can be effectively exploited to develop them as promising quadruplex-targeting agents. Herein, the design, synthesis and evaluation of a series of dimethylindolenine cyanine dyes with varying halogen substitutions are reported. Their interactions with telomeric and c-myc quadruplexes as well as a reference duplex sequence have been evaluated using thermal melting, biosensor-surface plasmon resonance, circular dichroism, isothermal titration calorimetry and mass spectrometry. Thermal melting analysis indicates that these ligands exhibit significant quadruplex stabilization and a very low duplex binding, with the dimethyl incorporation of paramount importance for decreased duplex affinity. Circular dichroism studies showed that the interaction of cyanines with quadruplex structures are primarily through stacking at one or both ends of the terminal tetrads with the two (trimethylammonium)propyl groups interacting in the accessible quadruplex grooves. Surface plasmon resonance and mass spectral studies shows the formation of an initial strong 1:1 complex followed by a significantly weaker secondary binding. Isothermal calorimetry studies show that the interaction of cyanines is predominantly entropy driven. In line with the design principles, this work provides new insights for further developing potent, highly selective cyanines as promising quadruplex-specific agents.

  14. Interactions between meso-tetrakis(4-(N-methylpyridiumyl))porphyrin TMPyP4 and DNA G-quadruplex of telomeric repeated sequence TTAGGG

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The binding properties between meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the parallel DNA G-quadruplex (G4) of telomeric repeated sequence 5′-TTAGGG-3′ have been characterized by means of circular dichroism,steady-state absorption,steady-state fluorescence and picosecond time-resolved fluorescence spectroscopies. The binding constant and the saturated binding number were determined as 1.29×106 (mol/L)-1 and 3,respectively,according to steady-state absorption spec-troscopy. Based on the findings by the use of time-resolved fluorescence spectroscopic technique,it is deduced that TMPyP4 binds to a DNA G-quadruplex with both the thread-intercalating and end-stacking modes and at the saturated binding state,one TMPyP4 molecule intercalates into the intervals of G-tetrads while the other two stack to the ends of the DNA G-quadruplex.

  15. Sequencing and G-quadruplex folding of the canine proto-oncogene KIT promoter region: might dog be used as a model for human disease?

    Directory of Open Access Journals (Sweden)

    Silvia Da Ros

    Full Text Available Downregulation of gene expression by induction of non-canonical DNA structures at promotorial level is a novel attractive anticancer strategy. In human, two guanine-rich sequences (h_kit1 and h_kit2 were identified in the promotorial region of oncogene KIT. Their stabilization into G-quadruplex structures can find applications in the treatment of leukemias, mastocytosis, gastrointestinal stromal tumor, and lung carcinomas which are often associated to c-kit mis-regulation. Also the most common skin cancer in domestic dog, mast cell tumor, is linked to a mutation and/or to an over-expression of c-kit, thus supporting dog as an excellent animal model. In order to assess if the G-quadruplex mediated mechanism of regulation of c-kit expression is conserved among the two species, herein we cloned and sequenced the canine KIT promoter region and we compared it with the human one in terms of sequence and conformational equilibria in physiologically relevant conditions. Our results evidenced a general conserved promotorial sequence between the two species. As experimentally confirmed, this grants that the conformational features of the canine kit1 sequence are substantially shared with the human one. Conversely, two isoforms of the kit2 sequences were identified in the analyzed dog population. In comparison with the human counterpart, both of them showed an altered distribution among several folded conformations.

  16. Sequencing and G-Quadruplex Folding of the Canine Proto-Oncogene KIT Promoter Region: Might Dog Be Used as a Model for Human Disease?

    Science.gov (United States)

    Da Ros, Silvia; Zorzan, Eleonora; Giantin, Mery; Zorro Shahidian, Lara; Palumbo, Manlio; Dacasto, Mauro; Sissi, Claudia

    2014-01-01

    Downregulation of gene expression by induction of non-canonical DNA structures at promotorial level is a novel attractive anticancer strategy. In human, two guanine-rich sequences (h_kit1 and h_kit2) were identified in the promotorial region of oncogene KIT. Their stabilization into G-quadruplex structures can find applications in the treatment of leukemias, mastocytosis, gastrointestinal stromal tumor, and lung carcinomas which are often associated to c-kit mis-regulation. Also the most common skin cancer in domestic dog, mast cell tumor, is linked to a mutation and/or to an over-expression of c-kit, thus supporting dog as an excellent animal model. In order to assess if the G-quadruplex mediated mechanism of regulation of c-kit expression is conserved among the two species, herein we cloned and sequenced the canine KIT promoter region and we compared it with the human one in terms of sequence and conformational equilibria in physiologically relevant conditions. Our results evidenced a general conserved promotorial sequence between the two species. As experimentally confirmed, this grants that the conformational features of the canine kit1 sequence are substantially shared with the human one. Conversely, two isoforms of the kit2 sequences were identified in the analyzed dog population. In comparison with the human counterpart, both of them showed an altered distribution among several folded conformations. PMID:25084283

  17. Blocking the binding of WT1 to bcl-2 promoter by G-quadruplex ligand SYUIQ-FM05

    Directory of Open Access Journals (Sweden)

    Yun-Xia Xiong

    2016-03-01

    Full Text Available At present, wt1, a Wilms’ tumor suppressor gene, is recognized as a critical regulator of tumorigenesis and a potential therapeutic target. WT1 shows the ability to regulate the transcription of bcl-2 by binding to a GC-rich region in the promoter, which can then fold into a special DNA secondary structure called the G-quadruplex. This function merits the exploration of the effect of a G-quadruplex ligand on the binding and subsequent regulation of WT1 on the bcl-2 promoter. In the present study, WT1 was found to bind to the double strand containing the G-quadruplex-forming sequence of the bcl-2 promoter. However, the G-quadruplex ligand SYUIQ-FM05 effectively blocked this binding by interacting with the GC-rich sequence. Our new findings are significant in the exploration of new strategies to block WT1's transcriptional regulation for cancer-cell treatment.

  18. Topological effects of charge transfer in telomere G-quadruplex: Mechanism on telomerase activation and inhibition

    CERN Document Server

    Wang, Xin

    2015-01-01

    We explore charge transfer in the telomere G-Quadruplex (TG4) DNA theoretically by the nonequilibrium Green's function method, and reveal the topological effect of charge transport in TG4 DNA. The consecutive TG4(CTG4) is semiconducting with 0.2 ~ 0.3eV energy gap. Charges transfers favorably in the consecutive TG4, but are trapped in the non-consecutive TG4 (NCTG4). The global conductance is inversely proportional to the local conductance for NCTG4. The topological structure transition from NCTG4 to CTG4 induces abruptly ~ 3nA charge current, which provide a microscopic clue to understand the telomerase activated or inhibited by TG4. Our findings reveal the fundamental property of charge transfer in TG4 and its relationship with the topological structure of TG4.

  19. Topological Effects of Charge Transfer in Telomere G-Quadruplex Mechanism on Telomerase Activation and Inhibition

    Science.gov (United States)

    Wang, Xin; Liang, Shi-Dong

    2013-02-01

    We explore the charge transfer in the telomere G-Quadruplex (TG4) DNA theoretically by the nonequilibrium Green's function method, and reveal the topological effect of the charge transport in TG4 DNA. The consecutive TG4 (CTG4) is semiconducting with 0.2 0.3 eV energy gap. Charges transfer favorably in the CTG4, but are trapped in the nonconsecutive TG4 (NCTG4). The global conductance is inversely proportional to the local conductance for NCTG4. The topological structure transition from NCTG4 to CTG4 induces abruptly 3nA charge current, which provide a microscopic clue to understand the telomerase activated or inhibited by TG4. Our findings reveal the fundamental property of charge transfer in TG4 and its relationship with the topological structure of TG4.

  20. Resolution of Quadruplex Polymorphism by Size Exclusion Chromatography

    Science.gov (United States)

    Miller, M. Clarke; Trent, John O.

    2014-01-01

    This unit describes a method for the separation of quadruplex species formed from the same sequence via size exclusion chromatography (SEC). Polymorphism is inherent to quadruplex formation, and even relatively simple quadruplex forming sequences, such as the human telomere sequence d(GGG(TTAGGG)3), can form a myriad of possible configurations. High Performance Liquid Chromatography (HPLC), especially reverse phase and anion exchange methods, has been a mainstay of nucleic acids research and purification for many decades. We have applied these methods for the separation of individual quadruplex species formed in a mixture from the same parent sequence. PMID:21638270

  1. Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration.

    Science.gov (United States)

    Gilbert-Girard, Shella; Gravel, Annie; Artusi, Sara; Richter, Sara N; Wallaschek, Nina; Kaufer, Benedikt B; Flamand, Louis

    2017-07-15

    Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6A/B integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevents telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex-specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on the HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. We next investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A's ability to integrate its genome into host chromosomes. Incubation of telomerase-expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency (P G-quadruplex. In the current study, we have examined the effects of a G-quadruplex binding and stabilizing agent, BRACO-19, on HHV-6A chromosomal integration. By stabilizing G-quadruplex structures, BRACO-19 affects the ability of the telomerase complex to elongate telomeres. Our results indicate that BRACO-19 reduces the number of clones harboring integrated HHV-6A. This study is the first of its kind and suggests that telomerase activity is essential to restore a functional telomere of adequate length following HHV-6A integration. Copyright © 2017 American Society for Microbiology.

  2. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins.

    Science.gov (United States)

    Foulk, Michael S; Urban, John M; Casella, Cinzia; Gerbi, Susan A

    2015-05-01

    Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (λ-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. We used genomics and biochemical approaches to determine if λ-exo digests all parental DNA sequences equally. We report that λ-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, λ-exo digestion of nonreplicating genomic DNA (LexoG0) enriches GC-rich DNA and G4 motifs genome-wide. We used LexoG0 data to control for nascent strand-independent λ-exo biases in NS-seq and validated this approach at the rDNA locus. The λ-exo-controlled NS-seq peaks are not GC-rich, and only 35.5% overlap with 6.8% of all G4s, suggesting that G4s are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na(+) instead of K(+) in the λ-exo digestion buffer reduced the effect of G4s on λ-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq.

  3. The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

    Science.gov (United States)

    Scalabrin, Matteo; Frasson, Ilaria; Ruggiero, Emanuela; Perrone, Rosalba; Tosoni, Elena; Lago, Sara; Tassinari, Martina; Palù, Giorgio; Richter, Sara N.

    2017-01-01

    G-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. A dynamic G-quadruplex forming region in the HIV-1 LTR promoter represses HIV-1 transcription when in the folded conformation. This activity is enhanced by nucleolin, which induces and stabilizes the HIV-1 LTR G-quadruplexes. In this work by a combined pull-down/mass spectrometry approach, we consistently found hnRNP A2/B1 as an additional LTR-G-quadruplex interacting protein. Surface plasmon resonance confirmed G-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnRNP A2/B1 is able to efficiently unfold the LTR G-quadruplexes. Evaluation of the thermal stability of the LTR G-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the LTR full-length environment. When hnRNP A2/B1 was silenced in cells, LTR activity decreased, indicating that the protein acts as a HIV-1 transcription activator. Our data highlight a tightly regulated control of transcription based on G-quadruplex folding/unfolding, which depends on interacting cellular proteins. These findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated HIV-1, present both in actively and latently infected cells.

  4. Factors influencing the performance of G-quadruplex DNAzyme-based sensors.

    Science.gov (United States)

    Kong, De-Ming

    2013-12-15

    G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Compared with natural enzymes, G-quadruplex DNAzyme offers many advantages, thus making it a promising tool in the design of biosensors and chemical sensors. Many biosensors and chemical sensors based on G-quadruplex DNAzymes have been reported. A number of factors may affect the performance of G-quadruplex DNAzyme-based sensors. Here we focus on some aspects to be taken into account when designing a G-quadruplex DNAzyme-based sensor. These include the G-quadruplex-forming G-rich sequence, solution components, the reaction substrate, and enrichment strategy for G-quadruplex DNAzyme. We also provide an outlook for further research on G-quadruplex DNAzyme-based sensors.

  5. Cation Coordination Alters the Conformation of a Thrombin-Binding G-Quadruplex DNA Aptamer That Affects Inhibition of Thrombin.

    Science.gov (United States)

    Zavyalova, Elena; Tagiltsev, Grigory; Reshetnikov, Roman; Arutyunyan, Alexander; Kopylov, Alexey

    2016-10-01

    Thrombin-binding aptamers are promising anticoagulants. HD1 is a monomolecular antiparallel G-quadruplex with two G-quartets linked by three loops. Aptamer-thrombin interactions are mediated with two TT-loops that bind thrombin exosite I. Several cations were shown to be coordinated inside the G-quadruplex, including K(+), Na(+), NH4(+), Ba(2+), and Sr(2+); on the contrary, Mn(2+) was coordinated in the grooves, outside the G-quadruplex. K(+) or Na(+) coordination provides aptamer functional activity. The effect of other cations on aptamer functional activity has not yet been described, because of a lack of relevant tests. Interactions between aptamer HD1 and a series of cations were studied. A previously developed enzymatic method was applied to evaluate aptamer inhibitory activity. The structure-function correlation was studied using the characterization of G-quadruplex conformation by circular dichroism spectroscopy. K(+) coordination provided the well-known high inhibitory activity of the aptamer, whereas Na(+) coordination supported low activity. Although NH4(+) coordination yielded a typical antiparallel G-quadruplex, no inhibitory activity was shown; a similar effect was observed for Ba(2+) and Sr(2+) coordination. Mn(2+) coordination destabilized the G-quadruplex that drastically diminished aptamer inhibitory activity. Therefore, G-quadruplex existence per se is insufficient for aptamer inhibitory activity. To elicit the nature of these effects, we thoroughly analyzed nuclear magnetic resonance (NMR) and X-ray data on the structure of the HD1 G-quadruplex with various cations. The most reasonable explanation is that cation coordination changes the conformation of TT-loops, affecting thrombin binding and inhibition. HD1 counterparts, aptamers 31-TBA and NU172, behaved similarly with some distinctions. In 31-TBA, an additional duplex module stabilized antiparallel G-quadruplex conformation at high concentrations of divalent cations; whereas in NU172, a

  6. Visual detection and differentiation of Classic Swine Fever Virus strains using nucleic acid sequence-based amplification (NASBA) and G-quadruplex DNAzyme assay

    Science.gov (United States)

    Lu, Xiaolu; Shi, Xueyao; Wu, Gege; Wu, Tiantian; Qin, Rui; Wang, Yi

    2017-01-01

    The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses. PMID:28287135

  7. Using G-quadruplex/hemin to "switch-on" the cathodic photocurrent of p-type PbS quantum dots: toward a versatile platform for photoelectrochemical aptasensing.

    Science.gov (United States)

    Wang, Guang-Li; Shu, Jun-Xian; Dong, Yu-Ming; Wu, Xiu-Ming; Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2015-03-03

    We present a novel photoelectrochemical (PEC) biosensing platform by taking advantage of the phenomenon that hemin intercalated in G-quadruplex "switched-on" the cathode photocurrent of p-type PbS quantum dots (QDs). Photoinduced electron transfer between PbS QDs and G-quadruplex/hemin(III) complexes with the subsequent catalytic oxygen reduction by the reduced G-quadruplex/hemin(II) led to an obvious enhancement in the cathodic photocurrent of the PbS QDs. For the detection process, in the presence of hemin, the specific recognition of the targets with the sensing sequence would trigger the formation of a stable G-quadruplex/hemin complex, which will result in reduced charge recombination and hence amplified photocurrent intensity of the PbS QDs. By using different target sequences, the developed system made possible a novel, label-free "switch-on" PEC aptasensor toward versatile biomolecular targets such as DNA and thrombin. Especially, with ambient oxygen to regenerate G-quadruplex/hemin(II) to G-quadruplex/hemin(III), this substrate-free strategy not only promoted the photoelectric effect and thus the enhanced sensitivity of the system, but also avoided the addition of supplementary substrates of G-quadruplex/hemin such as H2O2 and organic substances.

  8. Hemin/G-quadruplex structure and activity alteration induced by magnesium cations.

    Science.gov (United States)

    Kosman, J; Juskowiak, B

    2016-04-01

    The influence of metal cations on G-quadruplex structure and peroxidase-mimicking DNAzyme activity was investigated. Experiments revealed a significant role of magnesium ion, which in the presence of potassium cation influenced DNAzyme activity. This ability has been associated with alteration of G-quadruplex topology and consequently affinity to bind hemin molecule. It has been demonstrated that G-quadruplex based on PS2.M sequence under these conditions formed parallel topology, which exhibited lower activity than that observed in standard potassium-containing solution. On the other hand DNAzyme/magnesium ion system based on telomeric sequence, which did not undergo significant structural changes, exhibited higher peroxidase activity upon magnesium ion addition. In both cases, the stabilization effect of magnesium cations on G-quadruplex structure was observed. The mechanism of DNAzyme activity alteration by magnesium ion can be explained by its influence on the pKa value of DNAzyme. Magnesium ion decreased pKa for PS2.M based system but increased it for telomeric DNAzyme. Magnesium cation effect on G-quadruplex structure as well as DNAzyme activity is particularly important since this ion is one of the most common metal cations in biological samples.

  9. RHPS4 G-quadruplex ligand induces anti-proliferative effects in brain tumor cells.

    Science.gov (United States)

    Lagah, Sunil; Tan, I-Li; Radhakrishnan, Priya; Hirst, Robert A; Ward, Jennifer H; O'Callaghan, Chris; Smith, Stuart J; Stevens, Malcolm F G; Grundy, Richard G; Rahman, Ruman

    2014-01-01

    Telomeric 3' overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure. We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels. Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo. This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.

  10. G-Quadruplexes: Prediction, Characterization, and Biological Application.

    Science.gov (United States)

    Kwok, Chun Kit; Merrick, Catherine J

    2017-07-26

    Guanine (G)-rich sequences in nucleic acids can assemble into G-quadruplex structures that involve G-quartets linked by loop nucleotides. The structural and topological diversity of G-quadruplexes have attracted great attention for decades. Recent methodological advances have advanced the identification and characterization of G-quadruplexes in vivo as well as in vitro, and at a much higher resolution and throughput, which has greatly expanded our current understanding of G-quadruplex structure and function. Accumulating knowledge about the structural properties of G-quadruplexes has helped to design and develop a repertoire of molecular and chemical tools for biological applications. This review highlights how these exciting methods and findings have opened new doors to investigate the potential functions and applications of G-quadruplexes in basic and applied biosciences. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. RHPS4 G-quadruplex ligand induces anti-proliferative effects in brain tumor cells.

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    Sunil Lagah

    Full Text Available BACKGROUND: Telomeric 3' overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4, a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure. METHODS: We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4 and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels. RESULTS: Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo. CONCLUSION: This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.

  12. A triple stranded G-quadruplex formation in the promoter region of human myosin β(Myh7) gene.

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    Singh, Anju; Kukreti, Shrikant

    2017-09-19

    Regulatory regions in human genome, enriched in guanine-rich DNA sequences have the propensity to fold into G-quadruplex structures. On exploring the genome for search of G-tracts, it was interesting to find that promoter of Human Myosin Gene (MYH7) contains a conserved 23-mer G-rich sequence (HM-23). Mutations in this gene are associated with familial cardiomyopathy. Enrichment of MYH7 gene in G-rich sequences could possibly play a critical role in its regulation. We used polyacrylamide gel electrophoresis (PAGE), UV-Thermal denaturation (UV-Tm) and Circular Dichroism (CD), to demonstrate the formation of a G-quadruplex by 23-mer G-rich sequence HM23 in promoter location of MYH7 gene. We observed that the wild G-rich sequence HM23 containing consecutive G5 stretch in two stacks adopt G-quadruplexes of diverse molecularity by involvement of four-strand, three-strand and two-strands with same parallel topology. Interestingly, the mutated sequence in the absence of continuous G5 stretch obstructs the formation of three-stranded G-quadruplex. We demonstrated that continuous G5 stretch is mandatory for the formation of a unique three-stranded G-quadruplex. Presence of various transcription factors (TF) in vicinity of the sequence HM23 leave fair possibility of recognition by TF binding sites, and so modulate gene expression. These findings may add on our understanding about the effect of base change in the formation of varied structural species in similar solution condition. This study may give insight about structural polymorphism arising due to recognition of non-Watson-Crick G-quadruplex structures by cellular proteins and designing structure specific molecules.

  13. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae.

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    John A Capra

    2010-07-01

    Full Text Available G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs. Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

  14. Intramolecular telomeric G-quadruplexes dramatically inhibit DNA synthesis by replicative and translesion polymerases, revealing their potential to lead to genetic change.

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    Deanna N Edwards

    Full Text Available Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.

  15. Metal Cations in G-Quadruplex Folding and Stability

    Science.gov (United States)

    Bhattacharyya, Debmalya; Mirihana Arachchilage, Gayan; Basu, Soumitra

    2016-01-01

    This review is focused on the structural and physicochemical aspects of metal cation coordination to G-Quadruplexes (GQ) and their effects on GQ stability and conformation. G-quadruplex structures are non-canonical secondary structures formed by both DNA and RNA. G-quadruplexes regulate a wide range of important biochemical processes. Besides the sequence requirements, the coordination of monovalent cations in the GQ is essential for its formation and determines the stability and polymorphism of GQ structures. The nature, location, and dynamics of the cation coordination and their impact on the overall GQ stability are dependent on several factors such as the ionic radii, hydration energy, and the bonding strength to the O6 of guanines. The intracellular monovalent cation concentration and the localized ion concentrations determine the formation of GQs and can potentially dictate their regulatory roles. A wide range of biochemical and biophysical studies on an array of GQ enabling sequences have generated at a minimum the knowledge base that allows us to often predict the stability of GQs in the presence of the physiologically relevant metal ions, however, prediction of conformation of such GQs is still out of the realm. PMID:27668212

  16. Metal Cations in G-Quadruplex Folding and Stability

    Science.gov (United States)

    Bhattacharyya, Debmalya; Mirihana Arachchilage, Gayan; Basu, Soumitra

    2016-09-01

    This review is focused on the structural and physico-chemical aspects of metal cation coordination to G-Quadruplexes (GQ) and their effects on GQ stability and conformation. G-Quadruplex structures are non-canonical secondary structures formed by both DNA and RNA. G-quadruplexes regulate a wide range of important biochemical processes. Besides the sequence requirements, the coordination of monovalent cations in the GQ is essential for its formation and determines the stability and polymorphism of GQ structures. The nature, location and dynamics of the cation coordination and their impact on the overall GQ stability are dependent on several factors such as the ionic radii, hydration energy and the bonding strength to the O6 of guanines. The intracellular monovalent cation concentration and the localized ion concentrations determine the formation of GQs and can potentially dictate their regulatory roles. A wide range of biochemical and biophysical studies on an array of GQ enabling sequences have generated at a minimum the knowledge base that allows us to often predict the stability of GQs in presence of the physiologically relevant metal ions, however, prediction of conformation of such GQs is still out of the realm.

  17. Metal Cations in G-Quadruplex Folding and Stability

    Directory of Open Access Journals (Sweden)

    Debmalya Bhattacharyya

    2016-09-01

    Full Text Available This review is focused on the structural and physico-chemical aspects of metal cation coordination to G-Quadruplexes (GQ and their effects on GQ stability and conformation. G-Quadruplex structures are non-canonical secondary structures formed by both DNA and RNA. G-quadruplexes regulate a wide range of important biochemical processes. Besides the sequence requirements, the coordination of monovalent cations in the GQ is essential for its formation and determines the stability and polymorphism of GQ structures. The nature, location and dynamics of the cation coordination and their impact on the overall GQ stability are dependent on several factors such as the ionic radii, hydration energy and the bonding strength to the O6 of guanines. The intracellular monovalent cation concentration and the localized ion concentrations determine the formation of GQs and can potentially dictate their regulatory roles. A wide range of biochemical and biophysical studies on an array of GQ enabling sequences have generated at a minimum the knowledge base that allows us to often predict the stability of GQs in presence of the physiologically relevant metal ions, however, prediction of conformation of such GQs is still out of the realm.

  18. Quadruplex-single nucleotide polymorphisms (Quad-SNP) influence gene expression difference among individuals.

    Science.gov (United States)

    Baral, Aradhita; Kumar, Pankaj; Halder, Rashi; Mani, Prithvi; Yadav, Vinod Kumar; Singh, Ankita; Das, Swapan K; Chowdhury, Shantanu

    2012-05-01

    Non-canonical guanine quadruplex structures are not only predominant but also conserved among bacterial and mammalian promoters. Moreover recent findings directly implicate quadruplex structures in transcription. These argue for an intrinsic role of the structural motif and thereby posit that single nucleotide polymorphisms (SNP) that compromise the quadruplex architecture could influence function. To test this, we analysed SNPs within quadruplex motifs (Quad-SNP) and gene expression in 270 individuals across four populations (HapMap) representing more than 14,500 genotypes. Findings reveal significant association between quadruplex-SNPs and expression of the corresponding gene in individuals (P analysis of Quad-SNPs obtained from population-scale sequencing of 1000 human genomes showed relative selection bias against alteration of the structural motif. To directly test the quadruplex-SNP-transcription connection, we constructed a reporter system using the RPS3 promoter-remarkable difference in promoter activity in the 'quadruplex-destabilized' versus 'quadruplex-intact' promoter was noticed. As a further test, we incorporated a quadruplex motif or its disrupted counterpart within a synthetic promoter reporter construct. The quadruplex motif, and not the disrupted-motif, enhanced transcription in human cell lines of different origin. Together, these findings build direct support for quadruplex-mediated transcription and suggest quadruplex-SNPs may play significant role in mechanistically understanding variations in gene expression among individuals.

  19. G-quadruplex DNA biosensor for sensitive visible detection of genetically modified food.

    Science.gov (United States)

    Jiang, Xiaohua; Zhang, Huimin; Wu, Jun; Yang, Xiang; Shao, Jingwei; Lu, Yujing; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

    2014-10-01

    In this paper, a novel label-free G-quadruplex DNAzyme sensor has been proposed for colorimetric identification of GMO using CaMV 35S promoter sequence as the target. The binary probes can fold into G-quadruplex structure in the presence of DNA-T (Target DNA) and then combine with hemin to form a DNAzyme resembling horseradish peroxidase. The detection system consists of two G-rich probes with 2:2 split mode by using the absorbance and color of ABTS(2-) as signal reporter. Upon the addition of a target sequence, two probes both hybridize with target and then their G-rich sequences combine to form a G-quadruplex DNAzyme, and the DNAzyme can catalyze the reaction of ABTS(2-) with H2O2. Then the linear range is from 0.05 to 0.5 μM while detection limit is 5nM. These results demonstrate that the proposed G-quadruplex DNAzyme method could be used as a simple, sensitive and cost-effective approach for assays of GMO.

  20. Xanthene and Xanthone Derivatives as G-Quadruplex Stabilizing Ligands

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    Alessandro Altieri

    2013-10-01

    Full Text Available Following previous studies on anthraquinone and acridine-based G-quadruplex ligands, here we present a study of similar aromatic cores, with the specific aim of increasing G-quadruplex binding and selectivity with respect to duplex DNA. Synthesized compounds include two and three-side chain xanthone and xanthene derivatives, as well as a dimeric “bridged” form. ESI and FRET measurements suggest that all the studied molecules are good G-quadruplex ligands, both at telomeres and on G-quadruplex forming sequences of oncogene promoters. The dimeric compound and the three-side chain xanthone derivative have been shown to represent the best compounds emerging from the different series of ligands presented here, having also high selectivity for G-quadruplex structures with respect to duplex DNA. Molecular modeling simulations are in broad agreement with the experimental data.

  1. Ebola virus derived G-quadruplexes: Thiazole orange interaction.

    Science.gov (United States)

    Krafčíková, Petra; Demkovičová, Erika; Víglaský, Viktor

    2016-12-13

    The Ebola and Marburg viruses are some of the deadliest viruses in the world. In this study a series of G-rich DNA sequences derived from these types of viruses which possess the potential to form G-quadruplex structures are analyzed. A set of DNA oligonucleotides derived from original viral isolates was used as a representative modeling sequence with which to demonstrate the influence of thiazole orange on circular dichroism (CD) spectral profiles. The results show the unique profile of the induced CD (ICD) signal in the visible region caused by interactions between the ligand and G-quadruplexes. This ligand was found to stabilize the G-quadruplex structure and can also induce topological changes and facilitate G-quadruplex multimerization. Thus, the ICD signatures can be used to determine whether specific unknown sequences can form G-quadruplex motifs. The viral sequences were analyzed using standard spectral and electrophoretic methods. In addition, the ability to target G-quadruplexes located in filoviruses offers researchers attractive therapeutic targets which would be of particular use in the development of novel antiviral therapies. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

  2. Targeting C-myc G-Quadruplex: Dual Recognition by Aminosugar-Bisbenzimidazoles with Varying Linker Lengths

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    Nihar Ranjan

    2013-11-01

    Full Text Available G-quadruplexes are therapeutically important biological targets. In this report, we present biophysical studies of neomycin-Hoechst 33258 conjugates binding to a G-quadruplex derived from the C-myc promoter sequence. Our studies indicate that conjugation of neomycin to a G-quadruplex binder, Hoechst 33258, enhances its binding. The enhancement in G-quadruplex binding of these conjugates varies with the length and composition of the linkers joining the neomycin and Hoechst 33258 units.

  3. p53 binds human telomeric G-quadruplex in vitro.

    Science.gov (United States)

    Adámik, Matej; Kejnovská, Iva; Bažantová, Pavla; Petr, Marek; Renčiuk, Daniel; Vorlíčková, Michaela; Brázdová, Marie

    2016-01-01

    The tumor suppressor protein p53 is a key factor in genome stability and one of the most studied of DNA binding proteins. This is the first study on the interaction of wild-type p53 with guanine quadruplexes formed by the human telomere sequence. Using electromobility shift assay and ELISA, we show that p53 binding to telomeric G-quadruplexes increases with the number of telomeric repeats. Further, p53 strongly favors G-quadruplexes folded in potassium over those formed in sodium, thus indicating the telomeric G-quadruplex conformational selectivity of p53. The presence of the quadruplex-stabilizing ligand, N-methyl mesoporphyrin IX (NMM), increases p53 recognition of G-quadruplexes in potassium. Using deletion mutants and selective p53 core domain oxidation, both p53 DNA binding domains are shown to be crucial for telomeric G-quadruplex recognition.

  4. Anthracene-terpyridine metal complexes as new G-quadruplex DNA binders.

    Science.gov (United States)

    Gama, Sofia; Rodrigues, Inês; Mendes, Filipa; Santos, Isabel C; Gabano, Elisabetta; Klejevskaja, Beata; Gonzalez-Garcia, Jorge; Ravera, Mauro; Vilar, Ramon; Paulo, António

    2016-07-01

    The formation of quadruple-stranded DNA induced by planar metal complexes has particular interest in the development of novel anticancer drugs. This is especially relevant for the inhibition of telomerase, which plays an essential role in cancer cell immortalization and is overexpressed in ca. 85-90% of cancer cells. Moreover, G-quadruplexes also exist in other locations in the human genome, namely oncogene promoter regions, and it has been hypothesized that they play a regulatory role in gene transcription. Herein we report a series of new anthracene-containing terpyridine ligands and the corresponding Cu(II) and Pt(II) complexes, with different linkers between the anthracenyl moiety and the terpyridine chelating unit. The interaction of these ligands and metal complexes with different topologies of DNA was studied by several biophysical techniques. The Pt(II) and Cu(II) complexes tested showed affinity for quadruplex-forming sequences with a good selectivity over duplex DNA. Importantly, the free ligands do not have significant affinity for any of the DNA sequences used, which shows that the presence of the metal is essential for high affinity (and selectivity). This effect is more evident in the case of the Pt(II) complexes. Moreover, the presence of a longer linker between the chelating terpyridine unit and the anthracene moiety enhances the interaction with G-quadruplex-forming sequences. We further evaluated the ability of the Cu(II) complexes to interact with, and stabilize G-quadruplex containing regions in oncogene promoters via a polymerase stop assay. These studies indicated that the metal complexes are able to induce G-quadruplex formation and stop polymerase activity.

  5. Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models

    Science.gov (United States)

    Zorzan, Eleonora; Ros, Silvia Da; Musetti, Caterina; Shahidian, Lara Zorro; Ramos Coelho, Nuno Filipe; Bonsembiante, Federico; Létard, Sébastien; Gelain, Maria Elena; Palumbo, Manlio; Dubreuil, Patrice; Giantin, Mery; Sissi, Claudia; Dacasto, Mauro

    2016-01-01

    Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an “in house” library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters. From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT–dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., α155, HMC1.2 and ROSA cells). Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations. PMID:26942875

  6. An improved model for the hTERT promoter quadruplex.

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    Jonathan B Chaires

    Full Text Available Mutations occur at four specific sites in the hTERT promoter in >75% of glioblastomas and melanomas, but the mechanism by which the mutations affect gene expression remains unexplained. We report biophysical computational studies that show that the hTERT promoter sequence forms a novel G-quadruplex structure consisting of three contiguous, stacked parallel quadruplexes. The reported hTERT mutations map to the central quadruplex within this structure, and lead to an alteration of its hydrodynamic properties and stability.

  7. Spectroscopic, biological, and molecular modeling studies on the interactions of [Fe(III)-meloxicam] with G-quadruplex DNA and investigation of its release from bovine serum albumin (BSA) nanoparticles.

    Science.gov (United States)

    Ebrahimi, Malihe; Khayamian, Taghi; Hadadzadeh, Hassan; Sayed Tabatabaei, Badraldin Ebrahim; Jannesari, Zahra; Khaksar, Ghazale

    2015-01-01

    The guanine-rich sequence, specifically in DNA, telomeric DNA, is a potential target of anticancer drugs. In this work, a mononuclear Fe(III) complex containing two meloxicam ligands was synthesized as a G-quadruplex stabilizer. The interaction between the Fe(III) complex and G-quadruplex with sequence of 5'-G3(T2AG3)3-3' (HTG21) was investigated using spectroscopic methods, molecular modeling, and polymerase chain reaction (PCR) assays. The spectroscopic methods of UV-vis, fluorescence, and circular dichroism showed that the metal complex can effectively induce and stabilize G-quadruplex structure in the G-rich 21-mer sequence. Also, the binding constant between the Fe(III) complex and G-quadruplex was measured by these methods and it was found to be 4.53(±0.30) × 10(5) M(-1)). The PCR stop assay indicated that the Fe(III) complex inhibits DNA amplification. The cell viability assay showed that the complex has significant antitumor activities against Hela cells. According to the UV-vis results, the interaction of the Fe(III) complex with duplex DNA is an order of magnitude lower than G-quadruplex. Furthermore, the release of the complex incorporated in bovine serum albumin nanoparticles was also investigated in physiological conditions. The release of the complex followed a bi-phasic release pattern with high and low releasing rates at the first and second phases, respectively. Also, in order to obtain the binding mode of the Fe(III) complex with G-quadruplex, molecular modeling was performed. The molecular docking results showed that the Fe(III) complex was docked to the end-stacked of the G-quadruplex with a π-π interaction, created between the meloxicam ligand and the guanine bases of the G-quadruplex.

  8. Accurate high-throughput identification of parallel G-quadruplex topology by a new tetraaryl-substituted imidazole.

    Science.gov (United States)

    Hu, Ming-Hao; Chen, Shuo-Bin; Wang, Yu-Qing; Zeng, You-Mei; Ou, Tian-Miao; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2016-09-15

    G-quadruplex nucleic acids are four-stranded DNA or RNA secondary structures that are formed in guanine-rich sequences. These structures exhibit extensive structural polymorphism and play a pivotal role in the control of a variety of cellular processes. To date, diverse approaches for high-throughput identification of G-quadruplex structures have been successfully developed, but high-throughput methods for further characterization of their topologies are still lacking. In this study, we report a new tetra-arylimidazole probe psIZCM-1, which was found to display significant and distinctive changes in both the absorption and the fluorescence spectra in the presence of parallel G-quadruplexes but show insignificant changes upon interactions with anti-parallel G-quadruplexes or other non-quadruplex oligonucleotides. In view of this dual-output feature, we used psIZCM-1 to identify the parallel G-quadruplexes from a large set of 314 oligonucleotides (including 300 G-quadruplex-forming oligonucleotides and 14 non-quadruplex oligonucleotides) via a microplate reader and accordingly established a high-throughput method for the characterization of parallel G-quadruplex topologies. The accuracy of this method was greater than 95%, which was much higher than that of the commercial probe NMM. To make the approach more practical, we further combined psIZCM-1 with another G-quadruplex probe IZCM-7 to realize the high-throughput classification of parallel, anti-parallel G-quadruplexes and non-quadruplex structures.

  9. Aminoglycosylation can enhance the G-quadruplex binding activity of epigallocatechin.

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    Li-Ping Bai

    Full Text Available With the aim of enhancing G-quadruplex binding activity, two new glucosaminosides (16, 18 of penta-methylated epigallocatechin were synthesized by chemical glycosylation. Subsequent ESI-TOF-MS analysis demonstrated that these two glucosaminoside derivatives exhibit much stronger binding activity to human telomeric DNA and RNA G-quadruplexes than their parent structure (i.e., methylated EGC (14 as well as natural epigallocatechin (EGC, 6. The DNA G-quadruplex binding activity of 16 and 18 is even more potent than strong G-quadruplex binder quercetin, which has a more planar structure. These two synthetic compounds also showed a higher binding strength to human telomeric RNA G-quadruplex than its DNA counterpart. Analysis of the structure-activity relationship revealed that the more basic compound, 16, has a higher binding capacity with DNA and RNA G-quadruplexes than its N-acetyl derivative, 18, suggesting the importance of the basicity of the aminoglycoside for G-quadruplex binding activity. Molecular docking simulation predicted that the aromatic ring of 16 π-stacks with the aromatic ring of guanine nucleotides, with the glucosamine moiety residing in the groove of G-quadruplex. This research indicates that glycosylation of natural products with aminosugar can significantly enhance their G-quadruplex binding activities, thus is an effective way to generate small molecules targeting G-quadruplexes in nucleic acids. In addition, this is the first report that green tea catechin can bind to nucleic acid G-quadruplex structures.

  10. Enantioselective light switch effect of Δ- and Λ-[Ru(phenanthroline)2 dipyrido[3,2-a:2', 3'-c]phenazine](2+) bound to G-quadruplex DNA.

    Science.gov (United States)

    Park, Jin Ha; Lee, Hyun Suk; Jang, Myung Duk; Han, Sung Wook; Kim, Seog K; Lee, Young-Ae

    2017-07-04

    The interaction of Δ- and Λ-[Ru(phen)2DPPZ](2+) (DPPZ = dipyrido[3,2-a:2', 3'-c]phenazine, phen = phenanthroline) with a G-quadruplex formed from 5'-G2T2G2TGTG2T2G2-3'(15-mer) was investigated. The well-known enhancement of luminescence intensity (the 'light-switch' effect) was observed for the [Ru(phen)2DPPZ](2+) complexes upon formation of an adduct with the G-quadruplex. The emission intensity of the G-quadruplex-bound Λ-isomer was 3-fold larger than that of the Δ-isomer when bound to the G-quadruplex, which is opposite of the result observed in the case of double stranded DNA (dsDNA); the light switch effect is larger for the dsDNA-bound Δ-isomer. In the job plot of the G-quadruplex with Δ- and Λ-[Ru(phen)2DPPZ](2+), a major inflection point for the two isomers was observed at x ≈ .65, which suggests a binding stoichiometry of 2:1 for both enantiomers. When the G base at the 8th position was replaced with 6-methyl isoxanthopterin (6MI), a fluorescent guanine analog, the excited energy of 6-MI transferred to bound Δ- or Λ-[Ru(phen)2DPPZ](2+), which suggests that at least a part of both Ru(II) enantiomers is close to or in contact with the diagonal loop of the G-quadruplex. A luminescence quenching experiment using [Fe(CN)6](4-) for the G-quadruplex-bound Ru(II) complex revealed downward bending curves for both enantiomers in the Stern-Volmer plot, which suggests the presence of Ru(II) complexes that are both accessible and inaccessible to the quencher and may be related to the 2:1 binding stoichiometry.

  11. Irregular G-quadruplexes Found in the Untranslated Regions of Human mRNAs Influence Translation.

    Science.gov (United States)

    Bolduc, François; Garant, Jean-Michel; Allard, Félix; Perreault, Jean-Pierre

    2016-10-07

    G-quadruplex structures are composed of coplanar guanines and are found in both DNA and RNA. They are formed by the stacking of two or more G-quartets that are linked together by three loops. The current belief is that RNA G-quadruplexes include loops of l to 7 nucleotides in length, although recent evidence indicates that the central loop (loop 2) can be longer if loops 1 and 3 are limited to a single nucleotide each. With the objective of broadening the definition of irregular RNA G-quadruplexes, a bioinformatic search was performed to find potential G-quadruplexes located in the untranslated regions of human mRNAs (i.e. in the 5' and 3'-UTRs) that contain either a long loop 1 or 3 of up to 40 nucleotides in length. RNA molecules including the potential sequences were then synthesized and examined in vitro by in-line probing for the formation of G-quadruplex structures. The sequences that adopted a G-quadruplex structure were cloned into a luciferase dual vector and examined for their ability to modulate translation in cellulo Some irregular G-quadruplexes were observed to either promote or repress translation regardless of the position or the size of the long loop they possessed. Even if the composition of a RNA G-quadruplex is not quite completely understood, the results presented in this report clearly demonstrate that what defines a RNA G-quadruplex is much broader than what we previously believed.

  12. Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay.

    Science.gov (United States)

    Noureini, Sakineh Kazemi; Esmaeili, Hosein; Abachi, Farzane; Khiali, Soraia; Islam, Barira; Kuta, Martyna; Saboury, Ali A; Hoffmann, Marcin; Sponer, Jiri; Parkinson, Gary; Haider, Shozeb

    2017-08-01

    Natural bioproducts are invaluable resources in drug discovery. Isoquinoline alkaloids of Chelidonium majus constitute a structurally diverse family of natural products that are of great interest, one of them being their selectivity for human telomeric G-quadruplex structure and telomerase inhibition. The study focuses on the mechanism of telomerase inhibition by stabilization of telomeric G-quadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol (q-TRAP) and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric G-quadruplex forming sequences were explored using a sensitive modified thermal FRET-melting measurement in the presence of the complementary oligonucleotide CT22. We assessed and monitored G-quadruplex topologies using circular dichroism (CD) methods, and compared spectra to previously well-characterized motifs, either alone or in the presence of the alkaloids. Molecular modeling was performed to rationalize ligand binding to the G-quadruplex structure. The results highlight strong inhibitory effects of chelerythrine, sanguinarine and berberine on telomerase activity, most likely through substrate sequestration. These isoquinoline alkaloids interacted strongly with telomeric sequence G-quadruplex. In comparison, chelidonine and papaverine had no significant interaction with the telomeric quadruplex, while they strongly inhibited telomerase at transcription level of hTERT. Altogether, all of the studied alkaloids showed various levels and mechanisms of telomerase inhibition. We report on a comparative study of anti-telomerase activity of the isoquinoline alkaloids of Chelidonium majus. Chelerythrine was most effective in inhibiting telomerase activity by substrate sequesteration through G-quadruplex

  13. Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands.

    Science.gov (United States)

    Bay, Daniyah H; Busch, Annika; Lisdat, Fred; Iida, Keisuke; Ikebukuro, Kazunori; Nagasawa, Kazuo; Karube, Isao; Yoshida, Wataru

    2017-06-27

    G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay. When G-quadruplex-forming sequences from the Dele and Cdc6 genes have been cloned in reporter vectors carrying a minimal promoter and the luciferase gene, luciferase expression is activated. This has also been detected in experiments applying a promoterless reporter vector. Mutational analysis reveals that guanine bases, which form the G-tetrads, are important in the activation. In addition, the activation has been found to decrease by the telomestatin derivative L1H1-7OTD which can bind to the G-quadruplex DNA. When Dele and Cdc6 CpG islands, containing the G-quadruplex-forming sequence, have been cloned in the promoterless reporter vector, the luciferase expression is activated. Mutational analysis reveals that the expression level is decreased by mutation on Dele G-quadruplex; however, increased by mutation on Cdc6 G-quadruplex. Dele and Cdc6 G-quadruplex formation is significant in the transcriptional regulation. Dele and Cdc6 G-quadruplex DNA alone possess enhancer and promotor function. When studied in more complex CpG islands Dele G-quadruplex also demonstrates promotor activity, whereas Cdc6 G-quadruplex may possess a dual function of transcriptional regulation.

  14. The binding modes of carbazole derivatives with telomere G-quadruplex

    Science.gov (United States)

    Zhang, Xiu-feng; Zhang, Hui-juan; Xiang, Jun-feng; Li, Qian; Yang, Qian-fan; Shang, Qian; Zhang, Yan-xia; Tang, Ya-lin

    2010-10-01

    It is reported that carbazole derivatives can stabilize G-quadruplex DNA structure formed by human telomeric sequence, and therefore, they have the potential to serve as anti-cancer agents. In this present study, in order to further explore the binding mode between carbazole derivatives and G-quadruplex formed by human telomeric sequence, two carbazole iodides (BMVEC, MVEC) molecules were synthesized and used to investigate the interaction with the human telomeric parallel and antiparallel G-quadruplex structures by NMR, CD and molecular modeling study. Interestingly, it is the pivotal the cationic charge pendant groups of pyridinium rings of carbazole that plays an essential role in the stabilizing and binding mode of the human telomeric sequences G-quadruplex structure. It was found that BMVEC with two cationic charge pendant groups of pyridinium rings of 9-ethylcarbazole cannot only stabilize parallel G-quadruple of Hum6 by groove binding and G-tetrad stacking modes and antiparallel G-quadruplex of Hum22 by groove binding, but also induce the formation of mixed G-quadruplex of Hum22. While MVEC with one cationic charge pendant groups of pyridinium ring only can bind with the parallel G-quadruplex of Hum6 by the stacking onto the G4 G-tetrad and could not interact with the G-quadruplex of Hum22.

  15. Homologous PNA Hybridization to Noncanonical DNA G-Quadruplexes.

    Science.gov (United States)

    Kormuth, Karen A; Woolford, John L; Armitage, Bruce A

    2016-03-29

    Potential guanine (G) quadruplex-forming sequences (QFSs) found throughout the genomes and transcriptomes of organisms have emerged as biologically relevant structures. These G-quadruplexes represent novel opportunities for gene regulation at the DNA and RNA levels. Recently, the definition of functional QFSs has been expanding to include a variety of unconventional motifs, including relatively long loop sequences (i.e., >7 nucleotides) separating adjacent G-tracts. We have identified a QFS within the 25S rDNA gene from Saccharomyces cerevisae that features a long loop separating the two 3'-most G-tracts. An oligonucleotide based on this sequence, QFS3, folds into a stable G-quadruplex in vitro. We have studied the interaction between QFS3 and several loop mutants with a small, homologous (G-rich) peptide nucleic acid (PNA) oligomer that is designed to form a DNA/PNA heteroquadruplex. The PNA successfully invades the DNA quadruplex target to form a stable heteroquadruplex, but with surprisingly high PNA:DNA ratios based on surface plasmon resonance and mass spectrometric results. A model for high stoichiometry PNA-DNA heteroquadruplexes is proposed, and the implications for quadruplex targeting by G-rich PNA are discussed.

  16. Quadruplex forming promoter region of c-myc oncogene as a potential target for a telomerase inhibitory plant alkaloid, chelerythrine.

    Science.gov (United States)

    Ghosh, Saptaparni; Dasgupta, Dipak

    2015-03-27

    Guanine rich sequences present in the promoter region of oncogenes could fold into G-quadruplexes and modulate transcription. Equilibrium between folding and unfolding of the quadruplexes in these regions play important role in disease processes. We have studied the effect of a putative anticancer agent chelerythrine on G-rich NHE III1 present in the promoter region of c-myc oncogene. We have demonstrated the ability of chelerythrine, a telomerase inhibitor, to block the hybridization of Pu27 with its complementary strand via folding it into a quadruplex structure. Calorimetry shows that the association of Pu27 with chelerythrine is primarily enthalpy driven with high binding affinity (∼10(5) M(-1)). The association does not lead to any major structural perturbation of Pu27. The resulting 2:1 complex has enhanced stability as compared to free Pu27. Another notable feature is that the presence of molecular crowding agent like ficoll 70 does not change the mode of recognition though the binding affinity decreases. We suggest that the anticancer activity of chelerythrine could be ascribed to its ability to stabilize the quadruplex structure in the c-myc promoter region thereby downregulating its transcription. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. DNA quadruplex folding formalism--a tutorial on quadruplex topologies.

    Science.gov (United States)

    Karsisiotis, Andreas Ioannis; O'Kane, Christopher; Webba da Silva, Mateus

    2013-11-01

    Quadruplexes of DNA adopt a large variety of topologies that are dependent on their environment. We have been developing a formalism for quadruplex folding based on the relationship between base and its sugar--as defined by the glycosidic bond angle. By reducing the quadruplex stem to a description based on two finite states of the range of angles the glycosidic bond angle may adopt, the description of the relationships of type of loop and groove widths of a quadruplex stem are possible. In its current form this formalism has allowed for the prediction of some unimolecular quadruplex topologies. Its rules, whilst developed for unimolecular quadruplexes of three loops, are of general utility in understanding the interdependency of structural characteristics of multimolecular folds, as well as unimolecular quadruplexes of more than three loops. Here we describe current understanding of the interdependent structural features that define the quadruplex fold, and provide a tutorial for the use and application of this formalism. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. FANCJ promotes DNA synthesis through G-quadruplex structures

    NARCIS (Netherlands)

    Castillo Bosch, Pau; Segura-Bayona, Sandra; Koole, Wouter; van Heteren, Jane T; Dewar, James M; Tijsterman, Marcel; Knipscheer, Puck

    2014-01-01

    Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mut

  19. Macrocyclic G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, M C; Ulven, Trond

    2010-01-01

    G-quadruplex stabilizing compounds have recently received increased interest due to their potential application as anticancer therapeutics. A significant number of structurally diverse G-quadruplex ligands have been developed. Some of the most potent and selective ligands currently known are macr...

  20. Conformation Selective Antibody Enables Genome Profiling and Leads to Discovery of Parallel G-Quadruplex in Human Telomeres.

    Science.gov (United States)

    Liu, Hui-Yun; Zhao, Qi; Zhang, Tian-Peng; Wu, Yue; Xiong, Yun-Xia; Wang, Shi-Ke; Ge, Yuan-Long; He, Jin-Hui; Lv, Peng; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Gu, Lian-Quan; Ren, Jian; Zhao, Yong; Huang, Zhi-Shu

    2016-10-20

    G-quadruplexes are specialized secondary structures in nucleic acids that possess significant conformational polymorphisms. The precise G-quadruplex conformations in vivo and their relevance to biological functions remain controversial and unclear, especially for telomeric G-quadruplexes. Here, we report a novel single-chain variable fragment (scFv) antibody, D1, with high binding selectivity for parallel G-quadruplexes in vitro and in vivo. Genome-wide chromatin immunoprecipitation using D1 and deep-sequencing revealed the consensus sequence for parallel G-quadruplex formation, which is characterized by G-rich sequence with a short loop size (G-quadruplex was identified and its formation was regulated by small molecular ligands targeting and telomere replication. Together, parallel G-quadruplex specific antibody D1 was found to be a valuable tool for determination of G-quadruplex and its conformation, which will prompt further studies on the structure of G-quadruplex and its biological implication in vivo.

  1. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria

    Science.gov (United States)

    Guo, Junjie U.; Bartel, David P.

    2017-01-01

    In vitro, some RNAs can form stable four-stranded structures known as G-quadruplexes. Although RNA G-quadruplexes have been implicated in post-transcriptional gene regulation and diseases, direct evidence for their formation in cells has been lacking. Here, we identified thousands of mammalian RNA regions that can fold into G-quadruplexes in vitro, but in contrast to previous assumptions, these regions were overwhelmingly unfolded in cells. Model RNA G-quadruplexes that were unfolded in eukaryotic cells were folded when ectopically expressed in Escherichia coli; however, they impaired translation and growth, which helps explain why we detected few G-quadruplex–forming regions in bacterial transcriptomes. Our results suggest that eukaryotes have a robust machinery that globally unfolds RNA G-quadruplexes, whereas some bacteria have instead undergone evolutionary depletion of G-quadruplex–forming sequences. PMID:27708011

  2. Selective G-Quadruplex DNA Recognition by a New Class of Designed Cyanines

    Directory of Open Access Journals (Sweden)

    Markus W. Germann

    2013-11-01

    Full Text Available A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The results reveal that elevated quadruplex binding and specificity are highly sensitive to the polymethine chain length, heterocyclic structure and intrinsic charge of the compound. Biophysical experiments show that the compounds display significant selectivity for quadruplex binding with a higher preference for parallel stranded quadruplexes, such as cMYC. NMR studies revealed the primary binding through an end-stacking mode and SPR studies showed the strongest compounds have primary KD values below 100 nM that are nearly 100-fold weaker for duplexes. The high selectivity of these newly designed trimethine cyanines for quadruplexes as well as their ability to discriminate between different quadruplexes are extremely promising features to develop them as novel probes for targeting quadruplexes in vivo.

  3. Selective G-quadruplex DNA recognition by a new class of designed cyanines.

    Science.gov (United States)

    Nanjunda, Rupesh; Owens, Eric A; Mickelson, Leah; Dost, Tyler L; Stroeva, Ekaterina M; Huynh, Hang T; Germann, Markus W; Henary, Maged M; Wilson, W David

    2013-11-04

    A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The results reveal that elevated quadruplex binding and specificity are highly sensitive to the polymethine chain length, heterocyclic structure and intrinsic charge of the compound. Biophysical experiments show that the compounds display significant selectivity for quadruplex binding with a higher preference for parallel stranded quadruplexes, such as cMYC. NMR studies revealed the primary binding through an end-stacking mode and SPR studies showed the strongest compounds have primary KD values below 100 nM that are nearly 100-fold weaker for duplexes. The high selectivity of these newly designed trimethine cyanines for quadruplexes as well as their ability to discriminate between different quadruplexes are extremely promising features to develop them as novel probes for targeting quadruplexes in vivo.

  4. Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme.

    Science.gov (United States)

    Cheng, Mingpan; Zhou, Jun; Jia, Guoqing; Ai, Xuanjun; Mergny, Jean-Louis; Li, Can

    2017-08-01

    The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100mM K(+), loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. (1)D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Enhanced anti-HIV-1 activity of G-quadruplexes comprising locked nucleic acids and intercalating nucleic acids

    DEFF Research Database (Denmark)

    Pedersen, Erik Bjerregaard; Nielsen, Jakob Toudahl; Nielsen, Claus

    2011-01-01

    Two G-quadruplex forming sequences, 50-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylet......Two G-quadruplex forming sequences, 50-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4......-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming...

  6. G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents.

    Science.gov (United States)

    Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela

    2015-09-22

    Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented.

  7. Wild-type p53 binds to MYC promoter G-quadruplex

    Science.gov (United States)

    Petr, Marek; Helma, Robert; Polášková, Alena; Krejčí, Aneta; Dvořáková, Zuzana; Kejnovská, Iva; Navrátilová, Lucie; Adámik, Matej; Vorlíčková, Michaela; Brázdová, Marie

    2016-01-01

    G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320–393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes. PMID:27634752

  8. Wild-type p53 binds to MYC promoter G-quadruplex.

    Science.gov (United States)

    Petr, Marek; Helma, Robert; Polášková, Alena; Krejčí, Aneta; Dvořáková, Zuzana; Kejnovská, Iva; Navrátilová, Lucie; Adámik, Matej; Vorlíčková, Michaela; Brázdová, Marie

    2016-10-01

    G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320-393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes.

  9. Novel molecular targets for kRAS downregulation: promoter G-quadruplexes

    Science.gov (United States)

    2016-11-01

    quadruplex DNA and down-regulation of oncogene c-myc by quindoline derivatives. Journal of medicinal chemistry 50, 1465–1474 (2007). 9 Brown , R. V., Danford...KCl, the DMS cleavage pat - tern for the induced G-quadruplex in the WT sequence revealed a Fig. 2. Predominant G4 isoforms formedwithin the near kRAS...41 (2013) 4049–4064. [37] R.V. Brown , V.C. Gaerig, T. Simmons, T.A. Brooks, Helping Eve overcome ADAM: G-quadruplexes in the ADAM-15 promoter as new

  10. X-ray characterization of mesophases of human telomeric G-quadruplexes and other DNA analogues.

    Science.gov (United States)

    Yasar, Selcuk; Schimelman, Jacob B; Aksoyoglu, M Alphan; Steinmetz, Nicole F; French, Roger H; Parsegian, V Adrian; Podgornik, Rudolf

    2016-06-02

    Observed in the folds of guanine-rich oligonucleotides, non-canonical G-quadruplex structures are based on G-quartets formed by hydrogen bonding and cation-coordination of guanosines. In dilute 5'-guanosine monophosphate (GMP) solutions, G-quartets form by the self-assembly of four GMP nucleotides. We use x-ray diffraction to characterize the columnar liquid-crystalline mesophases in concentrated solutions of various model G-quadruplexes. We then probe the transitions between mesophases by varying the PEG solution osmotic pressure, thus mimicking in vivo molecular crowding conditions. Using the GMP-quadruplex, built by the stacking of G-quartets with no covalent linking between them, as the baseline, we report the liquid-crystalline phase behaviors of two other related G-quadruplexes: (i) the intramolecular parallel-stranded G-quadruplex formed by the 22-mer four-repeat human telomeric sequence AG3(TTAG3)3 and (ii) the intermolecular parallel-stranded G-quadruplex formed by the TG4T oligonucleotides. Finally, we compare the mesophases of the G-quadruplexes, under PEG-induced crowding conditions, with the corresponding mesophases of the canonical duplex and triplex DNA analogues.

  11. X-ray characterization of mesophases of human telomeric G-quadruplexes and other DNA analogues

    Science.gov (United States)

    Yasar, Selcuk; Schimelman, Jacob B.; Aksoyoglu, M. Alphan; Steinmetz, Nicole F.; French, Roger H.; Parsegian, V. Adrian; Podgornik, Rudolf

    2016-06-01

    Observed in the folds of guanine-rich oligonucleotides, non-canonical G-quadruplex structures are based on G-quartets formed by hydrogen bonding and cation-coordination of guanosines. In dilute 5‧-guanosine monophosphate (GMP) solutions, G-quartets form by the self-assembly of four GMP nucleotides. We use x-ray diffraction to characterize the columnar liquid-crystalline mesophases in concentrated solutions of various model G-quadruplexes. We then probe the transitions between mesophases by varying the PEG solution osmotic pressure, thus mimicking in vivo molecular crowding conditions. Using the GMP-quadruplex, built by the stacking of G-quartets with no covalent linking between them, as the baseline, we report the liquid-crystalline phase behaviors of two other related G-quadruplexes: (i) the intramolecular parallel-stranded G-quadruplex formed by the 22-mer four-repeat human telomeric sequence AG3(TTAG3)3 and (ii) the intermolecular parallel-stranded G-quadruplex formed by the TG4T oligonucleotides. Finally, we compare the mesophases of the G-quadruplexes, under PEG-induced crowding conditions, with the corresponding mesophases of the canonical duplex and triplex DNA analogues.

  12. Selective G-Quadruplex DNA Recognition by a New Class of Designed Cyanines

    OpenAIRE

    Germann, Markus W.; David Wilson, W.; Henary, Maged M.; Huynh, Hang T.; Stroeva, Ekaterina M.; Dost, Tyler L.; Rupesh Nanjunda; Leah Mickelson; Owens, Eric A.

    2013-01-01

    A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The ...

  13. Volumetric contributions of loop regions of G-quadruplex DNA to the formation of the tertiary structure.

    Science.gov (United States)

    Takahashi, Shuntaro; Sugimoto, Naoki

    2017-02-06

    DNA guanine-quadruplexes (G-quadruplexes) are unique DNA structures formed by guanine-rich sequences. The loop regions of G-quadruplexes play key roles in stability and topology of G-quadruplexes. Here, we investigated volumetric changes induced by pressure in the folding of the G-quadruplex formed by the thrombin binding aptamer (TBA) with mutations within the loop regions. The change of partial molar volume in the transition from coil to G-quadruplex, ∆Vtr, of TBA with a mutation from T to A in the 5' most loop (TBA T3A) was 75.5cm(3)mol(-1), which was larger than that of TBA (54.6cm(3)mol(-1)). TBA with a G to T mutation in the central loop (TBA G8T) had thermal stability similar to TBA T3A but a smaller ∆Vtr of 41.1cm(3)mol(-1). In the presence of poly(ethylene)glycol 200 (PEG200), ∆Vtr values were 14.7cm(3)mol(-1) for TBA T3A and 13.2cm(3)mol(-1) for TBA G8T. These results suggest that the two mutations destabilize the G-quadruplex structure differently. Thus, volumetric data obtained using pressure-based thermodynamic analyses provides information about the dynamics of the loop regions and the roles of loops in the stabilities and folding of G-quadruplex structures.

  14. Unprecedented right- and left-handed quadruplex structures formed by heterochiral oligodeoxyribonucleotides.

    Science.gov (United States)

    Virgilio, Antonella; Esposito, Veronica; Citarella, Giuseppe; Mangoni, Alfonso; Mayol, Luciano; Galeone, Aldo

    2011-07-01

    CD and NMR studies on heterochiral oligodeoxynucleotides (d/l-ODNs) forming quadruplex structures are reported. Heterochiral ODNs, based on sequence TGGGGT, are able to form stable either right- or left-handed quadruplexes depending on d/l ratio and residues position. Results suggest that the 3'-end and the core of the G-run are more important than the 5'-end in determining the quadruplex handness. Particularly, oligonucleotide T(D)G(D)G(L)G(L)G(D)T(D) (L34) at low temperatures forms a well-defined left-handed quadruplex, notwithstanding it is mostly composed by natural d residues. This structure is characterized by three all-anti G-tetrads and one all-syn G-tetrad.

  15. Cyclic ferrocenylnaphthalene diimide derivative as a new class of G-quadruplex DNA binding ligand.

    Science.gov (United States)

    Islam, Md Monirul; Sato, Shinobu; Shinozaki, Shingo; Takenaka, Shigeori

    2017-01-15

    To identify an effective ligand that binds to a G-quadruplex structure but not a double-stranded DNA (dsDNA), a set of biophysical and biochemical experiments were carried out using newly synthesized cyclic ferrocenylnaphthalene diimide (cFNDI, 1) or the non-cyclic derivative (2) with various structures of G-quadruplex DNAs and dsDNA. Compound 1 bound strongly to G-quadruplexes DNAs (10(6)M(-1) order) with diminished binding to dsDNA (10(4)M(-1) order) in 100mM AcOH-AcOK buffer (pH 5.5) containing 100mM KCl. Interestingly, 1 showed an approximately 50-fold higher selectivity to mixed hybrid-type telomeric G-quadruplex DNA (K=3.4×10(6)M(-1) and a 2:1 stoichiometry) than dsDNA (K=7.5×10(4)M(-1)) did. Furthermore, 1 showed higher thermal stability to G-quadruplex DNAs than it did to dsDNA with a preference for c-kit and c-myc G-quadruplex DNAs over telomeric and thrombin binding aptamers. Additionally, 1 exhibited telomerase inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.4μM. Compound 2 showed a preference for G-quadruplex; however, the binding affinity magnitude and preference were improved in 1 because the former had a cyclic structure.

  16. Cryptolepine Derivatives:Quadruplex-Interactive Agents as Inhibitors of Human Telomerase

    Institute of Scientific and Technical Information of China (English)

    HUANG,Zhi-Shu; ZHOU,Jin-Lin; LU,Yu-Jing; GU,Lian-Quan

    2004-01-01

    @@ Alkaloids are very important natural products. Most of them have biologic activity. Many novel drugs have been developed based on alkaloids, such as camptothecin, taxol, vinblastine. A series of novel cryptolepine derivatives were synthesized (Figure 1). The interaction of cryptolepine derivatives with G-quadruplex (Figure 2) was studied by CD and UV spectra.[1] Most of these compounds can induce the formation of G-quadruplex and stabilize the formed G-quadruplex, resulting in the inhibitory effect on telomerase. Most of these cryptolepine derivatives have potent cytotoxicity in vitro against human tumor cell line.

  17. G-quadruplexes as novel cis-elements controlling transcription during embryonic development.

    Science.gov (United States)

    David, Aldana P; Margarit, Ezequiel; Domizi, Pablo; Banchio, Claudia; Armas, Pablo; Calcaterra, Nora B

    2016-05-19

    G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.

  18. Chelerythrine down regulates expression of VEGFA, BCL2 and KRAS by arresting G-Quadruplex structures at their promoter regions

    Science.gov (United States)

    Jana, Jagannath; Mondal, Soma; Bhattacharjee, Payel; Sengupta, Pallabi; Roychowdhury, Tanaya; Saha, Pranay; Kundu, Pallob; Chatterjee, Subhrangsu

    2017-01-01

    A putative anticancer plant alkaloid, Chelerythrine binds to G-quadruplexes at promoters of VEGFA, BCL2 and KRAS genes and down regulates their expression. The association of Chelerythrine to G-quadruplex at the promoters of these oncogenes were monitored using UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy, CD melting, isothermal titration calorimetry, molecular dynamics simulation and quantitative RT-PCR technique. The pronounced hypochromism accompanied by red shifts in UV absorption spectroscopy in conjunction with ethidium bromide displacement assay indicates end stacking mode of interaction of Chelerythrine with the corresponding G-quadruplex structures. An increase in fluorescence anisotropy and CD melting temperature of Chelerythrine-quadruplex complex revealed the formation of stable Chelerythrine-quadruplex complex. Isothermal titration calorimetry data confirmed that Chelerythrine-quadruplex complex formation is thermodynamically favourable. Results of quantative RT-PCR experiment in combination with luciferase assay showed that Chelerythrine treatment to MCF7 breast cancer cells effectively down regulated transcript level of all three genes, suggesting that Chelerythrine efficiently binds to in cellulo quadruplex motifs. MD simulation provides the molecular picture showing interaction between Chelerythrine and G-quadruplex. Binding of Chelerythrine with BCL2, VEGFA and KRAS genes involved in evasion, angiogenesis and self sufficiency of cancer cells provides a new insight for the development of future therapeutics against cancer.

  19. Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes

    Directory of Open Access Journals (Sweden)

    Rowe J

    2009-08-01

    Full Text Available Abstract Background Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes. The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusion This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.

  20. Label-free detection of Cu(2+ and Hg(2+ ions using reconstructed Cu(2+-specific DNAzyme and G-quadruplex DNAzyme.

    Directory of Open Access Journals (Sweden)

    Hui Li

    Full Text Available Label-free metal ion detection methods were developed. To achieve these, a reconstructed Cu(2+-specific DNA-cleaving DNAzyme (Cu(2+-specific DNAzyme with an intramolecular stem-loop structure was used. G-quadruplex-forming G-rich sequence(s, linked at the ends of double-helix stem of an intramolecular stem-loop structure, was partly caged in an intramolecular duplex or formed a split G-quadruplex. Cu(2+-triggered DNA cleavage at a specific site decreased the stability of the double-helix stem, resulting in the formation or destruction of G-quadruplex DNAzyme that can effectively catalyze the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS-H2O2 reaction. Based on these, two label-free, cost-effective and simple Cu(2+ sensors were designed. These two sensors followed different detection modes: 'turn-on' and 'turn-off'. As for the 'turn-on' sensor, the intramolecular stem-loop structure ensured a low background signal, and the co-amplification of detection signal by dual DNAzymes (Cu(2+-specific DNAzyme and G-quadruplex DNAzyme provided a high sensitivity. This sensor enabled the selective detection of aqueous Cu(2+ with a detection limit of 3.9 nM. Visual detection was possible. Although the 'turn-off' sensor gave lower detection sensitivity than the 'turn-on' one, the characteristics of cost-effectiveness and ease of operation made it an important implement to reduce the possibility of pseudo-positive or pseudo-negative results. Combining the ability of Hg(2+ ion to stabilize T-T base mismatch, above dual DNAzymes-based strategy was further used for Hg(2+ sensor design. The proposed sensor allowed the specific detection of Hg(2+ ion with a detection of 4.8 nM. Visual detection was also possible.

  1. Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame

    Science.gov (United States)

    Rajendran, Arivazhagan; Endo, Masayuki; Hidaka, Kumi; Lan Thao Tran, Phong; Mergny, Jean-Louis; Sugiyama, Hiroshi

    2013-01-01

    Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex. PMID:23863846

  2. Thrombin-Binding Aptamer Quadruplex Formation: AFM and Voltammetric Characterization

    Directory of Open Access Journals (Sweden)

    Victor Constantin Diculescu

    2010-01-01

    Full Text Available The adsorption and the redox behaviour of thrombin-binding aptamer (TBA and extended TBA (eTBA were studied using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite and glassy carbon. The different adsorption patterns and degree of surface coverage were correlated with the sequence base composition, presence/absence of K+, and voltammetric behaviour of TBA and eTBA. In the presence of K+, only a few single-stranded sequences present adsorption, while the majority of the molecules forms stable and rigid quadruplexes with no adsorption. Both TBA and eTBA are oxidized and the only anodic peak corresponds to guanine oxidation. Upon addition of K+ ions, TBA and eTBA fold into a quadruplex, causing the decrease of guanine oxidation peak and occurrence of a new peak at a higher potential due to the oxidation of G-quartets. The higher oxidation potential of G-quartets is due to the greater difficulty of electron transfer from the inside of the quadruplex to the electrode surface than electron transfer from the more flexible single strands.

  3. A G-Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

    Institute of Scientific and Technical Information of China (English)

    邵从英; 鲁娜; 孙登明

    2012-01-01

    A heroin-binding DNA G-quadruplex (also known as a heroin aptamer or DNAzyme) has been previously re- ported to be able to enhance the peroxidase activity of heroin. In this work, we described a DNAzyme structure that had an effector-recognizing part appearing as a single stranded DNA linkage flanked by two split G-quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G-quadruplex halves and thus efficiently suppressed the enzymatic activity of the G-quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re-engineered G-quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G-quadruplex is an especially useful module to design low-cost and label-free sensors toward various biologically or environmentally interesting targets.

  4. Ruthenium Complex “Light Switches” that are Selective for Different G-Quadruplex Structures

    Science.gov (United States)

    Wachter, Erin; Moyá, Diego; Parkin, Sean

    2015-01-01

    Recognition and regulation of G-quadruplex nucleic acid structures is an important goal for the development of chemical tools and medicinal agents. The addition of a bromo substituent to the dipyridylphenazine (dppz) ligands in the photophysical “light switch”, [Ru(bpy)2dppz]2+, and the photochemical “light switch”, [Ru(bpy)2dmdppz]2+, creates compounds with increased selectivity for an intermolecular parallel G-quadruplex and the mixed-hybrid G-quadruplex, respectively. When [Ru(bpy)2dppz-Br]2+ and [Ru(bpy)2dmdppz-Br]2+ are incubated with the G-quadruplexes, they have a stabilizing effect on the DNA structures. Activation of [Ru(bpy)2dmdppz-Br]2+ with light results in covalent adduct formation with the DNA. These complexes demonstrate that subtle chemical modifications of RuII complexes can alter G-quadruplex selectivity, and could be useful for the rational design of in vivo G-quadruplex probes. PMID:26560887

  5. Behavior of the guanine base in G-quadruplexes probed by the fluorescent guanine analog, 6-methyl isozanthopterin

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ji Hoon; Chitrapriya, Nataraj; Lee, Hyun Suk; Lee, Young Ae; Kim, Seog K. [Dept. of Chemistry, Yeungnam University, Gyeongsan (Korea, Republic of); Jung, Maeng Joon [Dept. of Chemistry, Kyungpook National University, Daegu (Korea, Republic of)

    2017-02-15

    In this study, circular dichroism (CD) spectrum and fluorescence techniques were used to examine the dynamic properties and microenvironment of the guanine base (G) at the central loop and at the middle of the G-stem of the G-quadruplex formed from the G{sub 3}T{sub 2}G{sub 3}TGTG{sub 3}T{sub 2}G{sub 3} sequence (G-quadruplex 1), in which the G base at the 10th and 13th position were replaced with a fluorescent G analog, 6-methyl isoxanthopterin (6MI) (G-quadruplex 2 and 3, respectively). For all G-quadruplexes, the CD spectrum revealed a positive band at 263 nm and a shoulder at 298 nm, and the thermal melting profiles were the sum of at least two sigmoidal curves. These observations indicated the presence of two conformers in the G-quadruplex. The fluorescence intensity of G-quadruplex 2 was greater than 3, as expected from the extent of stacking interaction, which is larger in the G(6MI)G sequence than the T(6MI)T sequence. The efficiency of fluorescence quenching by the polar acrylamide quencher and negatively charged I− quencher were larger for G-quadruplex 3, suggesting that 6MI in the G(6MI)G stem is exposed more to the aqueous environment compared to that in the T(6MI)T central loop. In the latter case, 6MI may direct to the center of the top G-quartet layer. The possibility of hydrogen bond formation between the carbonyl group of 6MI and the acrylamide of the G-quadruplex 3 was proposed.

  6. Investigation of G-quadruplex formation in the FGFR2 promoter region and its transcriptional regulation by liensinine.

    Science.gov (United States)

    Zhang, Lulu; Tan, Wei; Zhou, Jiang; Xu, Ming; Yuan, Gu

    2017-04-01

    Fibroblast growth factor receptor 2 (FGFR2) is overexpressed in breast cancer tissues and cells, and has been shown to be a susceptibility factor for breast cancer. In this study, we found that the G-rich sequences in the FGFR2 promoter region can form G-quadruplexes, which could be the target and inhibitor of the FGFR2 gene. Initially, the formation of G-quadruplexes was confirmed by ESI-MS and CD, and DMS footprinting experiments gave the folding pattern of the G-quadruplexes. After luciferase reporter assays revealed that the G-quadruplex could inhibit the activity of the FGFR2 promoter, MS and SPR showed binding affinity and selectivity of the ligand. Then cell culture experiments and ChIP assay showed the bioactivity of the ligand. We found that three G-rich sequences (S1-S3) in the FGFR2 promoter region can form G-quadruplex structures. And a natural alkaloid, liensinine, was found to bind to the S1 G-quadruplex with relative high affinity and selectivity. Cell culture experiments showed that liensinine inhibits FGFR2 activity at both the transcriptional and translational levels. Moreover, chromatin immunoprecipitation assay (ChIP) results showed that liensinine blocks the binding of E2F1 at the transcription factor binding site (TFBS) in the S1 sequence, which is the mechanism through which liensinine inhibits the FGFR2 gene. A natural alkaloid was discovered to selectively bind to the S1 G-quadruplex with relative high affinity, and therefore inhibited FGFR2 transcription and translation. Our discovery offers a useful strategy to inhibit FGFR2 transcription, i.e., targeting the G-quadruplex with a natural alkaloid. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. G-Quadruplexes as Potential Therapeutic Targets for Embryonal Tumors

    Directory of Open Access Journals (Sweden)

    Michael Grotzer

    2013-10-01

    Full Text Available Embryonal tumors include a heterogeneous group of highly malignant neoplasms that primarily affect infants and children and are characterized by a high rate of mortality and treatment-related morbidity, hence improved therapies are clearly needed. G-quadruplexes are special secondary structures adopted in guanine (G-rich DNA sequences that are often present in biologically important regions, e.g. at the end of telomeres and in the regulatory regions of oncogenes such as MYC. Owing to the significant roles that both telomeres and MYC play in cancer cell biology, G-quadruplexes have been viewed as emerging therapeutic targets in oncology and as tools for novel anticancer drug design. Several compounds that target these structures have shown promising anticancer activity in tumor xenograft models and some of them have entered Phase II clinical trials. In this review we examine approaches to DNA targeted cancer therapy, summarize the recent developments of G-quadruplex ligands as anticancer drugs and speculate on the future direction of such structures as a potential novel therapeutic strategy for embryonal tumors of the nervous system.

  8. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins

    DEFF Research Database (Denmark)

    Foulk, M. S.; Urban, J. M.; Casella, Cinzia;

    2015-01-01

    Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (lambda-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent...... are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na+ instead of K...

  9. Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

    DEFF Research Database (Denmark)

    Krüger, Asger Christian; Raarup, Merete Krog; Nielsen, Morten Muhlig;

    2010-01-01

    G-rich telomeric DNA sequences can form G-quadruplex structures. The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and a shortened derivative (UP1) are active in telomere length regulation, and it has been reported that UP1 can unwind G-quadruplex structures. Here, we investigate...... the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G......-quadruplex telomeric DNA. Ensemble and single molecule FRET measurements provide further insight into molecular conformation: the telomeric DNA overhang is found to be in a folded state in the absence of hnRNP A1 and to remain predominantly in a compact state when complexed with hnRNP A1. This finding is in contrast...

  10. Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G-quadruplex aptamer and thrombin.

    Science.gov (United States)

    Wang, Jianhao; Gu, Yaqin; Liu, Li; Wang, Cheli; Wang, Jianpeng; Ding, Shumin; Li, Jinping; Qiu, Lin; Jiang, Pengju

    2017-08-01

    The dynamic binding status between the thrombin and its G-quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence-coupled capillary electrophoresis method. A 29-nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G-quadruplex from unstructured 29-nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6-carboxyfluorescein labeled G-quadruplex aptamer was measured. Third, the stability of G-quadruplex aptamer-thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29-nucleic acid thrombin binding aptamer could compete with G-quadruplex aptamer and thus disassociated the G-quadruplex structure into an unstructured aptamer. These data suggest that our in-house established fluorescence-coupled capillary electrophoresis assay could be applied to binding studies of the G-quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Formation of a unique cluster of G-quadruplex structures in the HIV-1 Nef coding region: implications for antiviral activity.

    Directory of Open Access Journals (Sweden)

    Rosalba Perrone

    Full Text Available G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment.

  12. Structure, Properties, and Biological Relevance of the DNA and RNA G-Quadruplexes: Overview 50 Years after Their Discovery.

    Science.gov (United States)

    Dolinnaya, N G; Ogloblina, A M; Yakubovskaya, M G

    2016-12-01

    G-quadruplexes (G4s), which are known to have important roles in regulation of key biological processes in both normal and pathological cells, are the most actively studied non-canonical structures of nucleic acids. In this review, we summarize the results of studies published in recent years that change significantly scientific views on various aspects of our understanding of quadruplexes. Modern notions on the polymorphism of DNA quadruplexes, on factors affecting thermodynamics and kinetics of G4 folding-unfolding, on structural organization of multiquadruplex systems, and on conformational features of RNA G4s and hybrid DNA-RNA G4s are discussed. Here we report the data on location of G4 sequence motifs in the genomes of eukaryotes, bacteria, and viruses, characterize G4-specific small-molecule ligands and proteins, as well as the mechanisms of their interactions with quadruplexes. New information on the structure and stability of G4s in telomeric DNA and oncogene promoters is discussed as well as proof being provided on the occurrence of G-quadruplexes in cells. Prominence is given to novel experimental techniques (single molecule manipulations, optical and magnetic tweezers, original chemical approaches, G4 detection in situ, in-cell NMR spectroscopy) that facilitate breakthroughs in the investigation of the structure and functions of G-quadruplexes.

  13. Fluorescence intercalator displacement assay for screening G4 ligands towards a variety of G-quadruplex structures.

    Science.gov (United States)

    Tran, Phong Lan Thao; Largy, Eric; Hamon, Florian; Teulade-Fichou, Marie-Paule; Mergny, Jean-Louis

    2011-08-01

    The potential formation of G-quadruplexes in many regions of the genome makes them an attractive target for drug design. A large number of small molecules synthesized in recent years display an ability to selectively target and stabilize G-quadruplexes. To screen for G4 ligands, we modified a G4-FID (G-quadruplex Fluorescent Intercalator Displacement) assay. This test is based on the displacement of an "on/off" fluorescence probe, Thiazole Orange (TO), from quadruplex or duplex DNA matrices by increasing amounts of a putative ligand. Selectivity measurements can easily be achieved by comparing the ability of the ligand to displace TO from various quadruplex and duplex structures. G4-FID requires neither modified oligonucleotides nor specific equipment and is an isothermal experiment. This test was adapted for high throughput screening onto 96-well plates allowing the comparison of more than twenty different structures. Fifteen different known G4 ligands belonging to different families were tested. Most compounds showed a good G4 vs duplex selectivity but exhibited little, if any, specificity for one quadruplex sequence over the others. The quest for the "perfect" specific G4 ligand is not over yet! Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  14. Interaction of Pyrrolobenzodiazepine (PBD) Ligands with Parallel Intermolecular G-Quadruplex Complex Using Spectroscopy and ESI-MS

    Science.gov (United States)

    Raju, Gajjela; Srinivas, Ragampeta; Santhosh Reddy, Vangala; Idris, Mohammed M.; Kamal, Ahmed; Nagesh, Narayana

    2012-01-01

    Studies on ligand interaction with quadruplex DNA, and their role in stabilizing the complex at concentration prevailing under physiological condition, has attained high interest. Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the interaction of PBD and TMPyP4 ligands, stoichiometry and selectivity to G-quadruplex DNA. Two synthetic ligands from PBD family, namely pyrene-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrid (PBD1), mixed imine-amide pyrrolobenzodiazepine dimer (PBD2) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) were studied. G-rich single-stranded oligonucleotide d(5′GGGGTTGGGG3′) designated as d(T2G8), from the telomeric region of Tetrahymena Glaucoma, was considered for the interaction with ligands. ESI-MS and spectroscopic methods viz., circular dichroism (CD), UV-Visible, and fluorescence were employed to investigate the G-quadruplex structures formed by d(T2G8) sequence and its interaction with PBD and TMPyP4 ligands. From ESI-MS spectra, it is evident that the majority of quadruplexes exist as d(T2G8)2 and d(T2G8)4 forms possessing two to ten cations in the centre, thereby stabilizing the complex. CD band of PBD1 and PBD2 showed hypo and hyperchromicity, on interaction with quadruplex DNA, indicating unfolding and stabilization of quadruplex DNA complex, respectively. UV-Visible and fluorescence experiments suggest that PBD1 bind externally where as PBD2 intercalate moderately and bind externally to G-quadruplex DNA. Further, melting experiments using SYBR Green indicate that PBD1 unfolds and PBD2 stabilizes the G-quadruplex complex. ITC experiments using d(T2G8) quadruplex with PBD ligands reveal that PBD1 and PBD2 prefer external/loop binding and external/intercalative binding to quadruplex DNA, respectively. From experimental results it is clear that the interaction of PBD2 and TMPyP4 impart higher stability to the quadruplex complex. PMID:22558271

  15. Steady-State and Time-Resolved Studies into the Origin of the Intrinsic Fluorescence of G-Quadruplexes.

    Science.gov (United States)

    Sherlock, Madeline E; Rumble, Christopher A; Kwok, Chun Kit; Breffke, Jens; Maroncelli, Mark; Bevilacqua, Philip C

    2016-06-16

    Stretches of guanines in DNA and RNA can fold into guanine quadruplex structures (GQSs). These structures protect telomeres in DNA and regulate gene expression in RNA. GQSs have an intrinsic fluorescence that is sensitive to different parameters, including loop sequence and length. However, the dependence of GQS fluorescence on solution and sequence parameters and the origin of this fluorescence are poorly understood. Herein we examine effects of dangling nucleotides and cosolute conditions on GQS fluorescence using both steady-state and time-resolved fluorescence spectroscopy. The quantum yield of dGGGTGGGTGGGTGGG, termed "dG3T", is found to be modest at ∼2 × 10(-3). Nevertheless, dG3T and its variants are significantly brighter than the common nucleic acid fluorophore 2-aminopurine (2AP) largely due to their sizable extinction coefficients. Dangling 5'-end nucleotides generally reduce emission and blue-shift the resultant spectrum, whereas dangling 3'-end nucleotides slightly enhance fluorescence, particularly on the red side of the emission band. Time-resolved fluorescence decays are broadly distributed in time and require three exponential components for accurate fits. Time-resolved emission spectra suggest the presence of two emitting populations centered at ∼330 and ∼390 nm, with the redder component being a well-defined long-lived (∼1 ns) entity. Insights into GQS fluorescence obtained here should be useful in designing brighter intrinsic RNA and DNA quadruplexes for use in label-free biotechnological applications.

  16. An isothermal titration and differential scanning calorimetry study of the G-quadruplex DNA-insulin interaction.

    Science.gov (United States)

    Timmer, Christine M; Michmerhuizen, Nicole L; Witte, Amanda B; Van Winkle, Margaret; Zhou, Dejian; Sinniah, Kumar

    2014-02-20

    The binding of insulin to the G-quadruplexes formed by the consensus sequence of the insulin-linked polymorphic region (ILPR) was investigated with differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The thermal denaturation temperature of insulin was increased by almost 4 °C upon binding to ILPR G-quadruplex DNA as determined by DSC. The thermodynamic parameters (K(D), ΔH, ΔG, and ΔS) of the insulin-G-quadruplex complex were further investigated by temperature-dependent ITC measurement over the range of 10-37 °C. The binding of insulin to the ILPR consensus sequence displays micromolar affinity in phosphate buffer at pH 7.4, which is mainly driven by entropic factors below 25 °C but by enthalpic terms above 30 °C. The interaction was also examined in several different buffers, and results showed that the observed ΔH is dependent on the ionization enthalpy of the buffer used. This indicates proton release upon the binding of G-quadruplex DNA to insulin. Additionally, the large negative change in heat capacity for this interaction may be associated with the dominant hydrophobicity of the amino acid sequence of insulin's β subunit, which is known to bind to the ILPR G-quadruplex DNA.

  17. Effect of Base Sequence on G-Wire Formation in Solution

    Directory of Open Access Journals (Sweden)

    Lea Spindler

    2010-01-01

    Full Text Available The formation and dimensions of G-wires by different short G-rich DNA sequences in solution were investigated by dynamic light scattering (DLS and polyacrilamide gel electrophoresis (PAGE. To explore the basic principles of wire formation, we studied the effects of base sequence, method of preparation, temperature, and oligonucleotide concentration. Both DLS and PAGE show that thermal annealing induces much less macromolecular self-assembly than dialysis. The degree of assembly and consequently length of G-wires (5-6 nm are well resolved by both methods for DNA sequences with intermediate length, while some discrepancies appear for the shortest and longest sequences. As expected, the longest DNA sequence gives the longest macromolecular aggregates with a length of about 11 nm as estimated by DLS. The quadruplex topologies show no concentration dependence in the investigated DNA concentration range (0.1 mM–0.4 mM and no structural change upon heating.

  18. Selective Targeting of G-Quadruplex Structures by a Benzothiazole-Based Binding Motif.

    Science.gov (United States)

    Buchholz, Ina; Karg, Beatrice; Dickerhoff, Jonathan; Sievers-Engler, Adrian; Lämmerhofer, Michael; Weisz, Klaus

    2017-03-09

    A benzothiazole derivative was identified as potent ligand for DNA G-quadruplex structures. Fluorescence titrations revealed selective binding to quadruplexes of different topologies including parallel, antiparallel and (3+1) hybrid structures. The parallel c-MYC sequence was found to constitute the preferred target with dissociation constants in the micromolar range. Binding of the benzothiazole-based ligand to c-MYC was structurally and thermodynamically characterized in detail by employing a comprehensive set of spectroscopic and calorimetric techniques. Job plot analyses and mass spectral data indicate non-cooperative ligand binding to form 1:1 and 2:1 complex stoichiometries. Whereas stacking interactions are suggested by optical methods, NMR chemical shift perturbations also indicate significant rearrangements of both 5'- and 3'-flanking sequences upon ligand binding. Additional isothermal calorimetry studies yield a thermodynamic profile of the ligand-quadruplex association and reveal enthalpic contributions to be the major driving force for binding. The structural and thermodynamic information obtained in the present work provides the basis for the rational development of benzothiazole derivatives as promising quadruplex binding agents.

  19. Small-angle X-ray scattering study of self-assembling lipophilic guanines in organic solvents: G-quadruplex formation and cation effects in cyclohexane.

    Science.gov (United States)

    Gonnelli, A; Ortore, M G; Baldassarri, E J; Spada, G P; Pieraccini, S; Perone, R C; Funari, S S; Mariani, P

    2013-01-31

    Lipophilic guanilic derivatives (lipoGs) dissolved in organic solvents can undergo different self-assembly pathways based on different H-bonded motifs, e.g., the cyclic discrete G-quartet, which forms in the presence of alkali-metal ions, and the "infinite" tape-like G-ribbon observed in the absence of ions. Using in-solution small-angle X-ray scattering, we analyzed a series of lipoGs dissolved in cyclohexane in the presence of different salts. The formation of G-quartet based supramolecular aggregates has been confirmed, evidencing the coexistence equilibrium of octamers and noncovalent molecular nanowires (the so-called G-quadruplexes). By global fitting the scattering data, the concentration of the two kinds of particles as well as the nanowire length have been derived as a function of temperature for the different compounds and salts. The thermodynamic parameters show that the self-assembly aggregation process is enthalpy driven, while the observed enthalpy-entropy compensation suggests that similar stacking interactions control the self-assembly of the different compounds. However, the strength of the stacking interactions, and then the nanowire stability, depends on the nature of templating cations and on their capacity to fill the central cavity of quadruplexes, with the order Sr(+) < Na(+) ≲ K(+).

  20. G-Quadruplex DNAzyme Molecular Beacon for Amplified Colorimetric Biosensing of Pseudostellaria heterophylla

    Directory of Open Access Journals (Sweden)

    Juan Hu

    2013-01-01

    Full Text Available With an internal transcribed spacer of 18 S, 5.8 S and 26 S nuclear ribosomal DNA (nrDNA ITS as DNA marker, we report a colorimetric approach for authentication of Pseudostellaria heterophylla (PH and its counterfeit species based on the differentiation of the nrDNA ITS sequence. The assay possesses an unlabelled G-quadruplex DNAzyme molecular beacon (MB probe, employing complementary sequence as biorecognition element and 1:1:1:1 split G-quadruplex halves as reporter. In the absence of target DNA (T-DNA, the probe can shape intermolecular G-quadruplex structures capable of binding hemin to form G-quadruplex-hemin DNAzyme and catalyze the oxidation of ABTS2− to blue-green ABTS•− by H2O2. In the presence of T-DNA, T-DNA can hybridize with the complementary sequence to form a duplex structure, hindering the formation of the G-quadruplex structure and resulting in the loss of the catalytic activity. Consequently, a UV-Vis absorption signal decrease is observed in the ABTS2−-H2O2 system. The “turn-off” assay allows the detection of T-DNA from 1.0 × 10−9 to 3.0 × 10−7 mol·L−1 (R2 = 0.9906, with a low detection limit of 3.1 × 10−10 mol·L−1. The present study provides a sensitive and selective method and may serve as a foundation of utilizing the DNAzyme MB sensor for identifying traditional Chinese medicines.

  1. A Novel Ultrasensitive ECL Sensor for DNA Detection Based on Nicking Endonuclease-Assisted Target Recycling Amplification, Rolling Circle Amplification and Hemin/G-Quadruplex

    Directory of Open Access Journals (Sweden)

    Fukang Luo

    2015-01-01

    Full Text Available In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease-assisted target recycling amplification (TRA, rolling circle amplification (RCA and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form “Y” junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O82− system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.

  2. G-quadruplex enhanced fluorescence of DNA-silver nanoclusters and their application in bioimaging

    Science.gov (United States)

    Zhu, Jinbo; Zhang, Libing; Teng, Ye; Lou, Baohua; Jia, Xiaofang; Gu, Xiaoxiao; Wang, Erkang

    2015-07-01

    Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially AS1411. Benefiting from the high brightness of AS1411 enhanced DNA-AgNCs and the specific binding affinity of AS1411 for nucleolin, the AS1411 enhanced AgNCs can stain cancer cells for bioimaging.Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially

  3. The G-quadruplex augments translation in the 5' untranslated region of transforming growth factor β2.

    Science.gov (United States)

    Agarwala, Prachi; Pandey, Satyaprakash; Mapa, Koyeli; Maiti, Souvik

    2013-03-05

    Transforming growth factor β2 (TGFβ2) is a versatile cytokine with a prominent role in cell migration, invasion, cellular development, and immunomodulation. TGFβ2 promotes the malignancy of tumors by inducing epithelial-mesenchymal transition, angiogenesis, and immunosuppression. As it is well-documented that nucleic acid secondary structure can regulate gene expression, we assessed whether any secondary motif regulates its expression at the post-transcriptional level. Bioinformatics analysis predicts an existence of a 23-nucleotide putative G-quadruplex sequence (PG4) in the 5' untranslated region (UTR) of TGFβ2 mRNA. The ability of this stretch of sequence to form a highly stable, intramolecular parallel quadruplex was demonstrated using ultraviolet and circular dichroism spectroscopy. Footprinting studies further validated its existence in the presence of a neighboring nucleotide sequence. Following structural characterization, we evaluated the biological relevance of this secondary motif using a dual luciferase assay. Although PG4 inhibits the expression of the reporter gene, its presence in the context of the entire 5' UTR sequence interestingly enhances gene expression. Mutation or removal of the G-quadruplex sequence from the 5' UTR of the gene diminished the level of expression of this gene at the translational level. Thus, here we highlight an activating role of the G-quadruplex in modulating gene expression of TGFβ2 at the translational level and its potential to be used as a target for the development of therapeutics against cancer.

  4. Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP).

    Science.gov (United States)

    Yang, Sunny Y; Amor, Souheila; Laguerre, Aurélien; Wong, Judy M Y; Monchaud, David

    2016-12-10

    The development of quadruplex-directed molecular diagnostic and therapy rely on mechanistic insights gained at both cellular and tissue levels by fluorescence imaging. This technique is based on fluorescent reporters that label cellular DNA and RNA quadruplexes to spatiotemporally address their complex cell biology. The photophysical characteristics of quadruplex probes usually dictate the modality of cell imaging by governing the selection of the light source (lamp, LED, laser), the optical light filters and the detection modality. Here, we report the characterizations of prototype from a new generation of quadruplex dye termed G4-REP (for quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality (owing to the versatility gained through the red-edge effect), thus allowing for diverse applications and most imaging facilities. This is demonstrated by cell images (and associated quantifications) collected through confocal and multiphoton microscopy as well as through real-time live-cell imaging system over extended period, monitoring both non-cancerous and cancerous human cell lines. Our results promote a new way of designing versatile, efficient and convenient quadruplex-reporting dyes for tracking these higher-order nucleic acid structures in living human cells. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

  5. Synthesis and Evaluation of a Rationally Designed Click-Based Library for G-Quadruplex Selective DNA Photocleavage

    Directory of Open Access Journals (Sweden)

    Dominic McBrayer

    2015-09-01

    Full Text Available DNA containing repeating G-rich sequences can adopt higher-order structures known as G-quadruplexes (G4. These structures are believed to form within telomeres and the promoter regions of some genes, particularly in a number of proto-oncogenes, where they may play a role in regulating transcription. Alternatively, G4 DNA may act as a barrier to replication. To investigate these potential biological roles, probes that combine highly selective G4 DNA targeting with photocleavage activity can allow temporal detection of G4 DNA, providing opportunities to obtain novel insights about the biological roles of G4 DNA. We have designed, synthesized, and screened a small library of potential selective G-quadruplex DNA photocleavage agents incorporating the G-quadruplex targeting moiety of 360A with known photocleavage groups linked via “click” chemistry.

  6. Modular Assembly of Cell-targeting Devices Based on an Uncommon G-quadruplex Aptamer

    DEFF Research Database (Denmark)

    Opazo, Felipe; Eiden, Laura; Hansen, Line

    2015-01-01

    Aptamers are valuable tools that provide great potential to develop cost-effective diagnostics and therapies in the biomedical field. Here, we report a novel DNA aptamer that folds into an unconventional G-quadruplex structure able to recognize and enter specifically into human Burkitt's lymphoma...

  7. Measurement of the base number of DNA using a special calliper made of a split G-quadruplex.

    Science.gov (United States)

    Zhu, Jinbo; Zhang, Libing; Wang, Erkang

    2012-12-21

    A special DNA calliper made of a split G-quadruplex was constructed to measure the length of DNA in a single-digit base number range between two selected sequences. The enhanced fluorescence of PPIX changed with the distance between two G-rich segments.

  8. Guanine quadruplex structures localize to heterochromatin

    NARCIS (Netherlands)

    R.F. Hoffmann; Y.M. Moshkin (Yuri); Mouton, S. (Stijn); Grzeschik, N.A. (Nicola A.); R.D. Kalicharan (Ruby); J. Kuipers (Jeroen); Wolters, A.H.G. (Anouk H.G.); Nishida, K. (Kazuki); A.V. Romashchenko; Postberg, J. (Jan); Lipps, H. (Hans); E. Berezikov (Eugene); O.C.M. Sibon (Ody); B.N.G. Giepmans (Ben); Lansdorp, P.M. (Peter M.)

    2016-01-01

    textabstractIncreasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for G4 DNA. Strikingly, immuno-electron microscopy showed exquisite specificity for heterochrom

  9. Guanine quadruplex structures localize to heterochromatin

    NARCIS (Netherlands)

    Hoffmann, Roland F.; Moshkin, Yuri M.; Mouton, Stijn; Grzeschik, Nicola A.; Kalicharan, Ruby D.; Kuipers, Jeroen; Wolters, Anouk H. G.; Nishida, Kazuki; Romashchenko, Aleksander V.; Postberg, Jan; Lipps, Hans; Berezikov, Eugene; Sibon, Ody C. M.; Giepmans, Ben N. G.; Lansdorp, Peter M.

    2016-01-01

    Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for G4 DNA. Strikingly, immuno-electron microscopy showed exquisite specificity for heterochromatin.

  10. Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds

    OpenAIRE

    Zimmer, J.; Tacconi, EM; Folio, C; Badie, S; Porru, M.; Klare, K; Tumiati, M; Markkanen, E; Halder, S.; Ryan, A; Jackson, SP; Ramadan, K; Kuznetsov, SG; Biroccio, A.; Sale, JE

    2015-01-01

    This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.molcel.2015.12.004 G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demons...

  11. Circular dichroism spectroscopic studies on structures formed by telomeric DNA sequences in vitro

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Telomere plays an important role in cellular processes, such as cell aging, death and carcinogenisis. Having special sequences, it can form quadruplex structure in vitro. Circular dichroism (CD) spectroscopic studies show that TTAGGG, (TTAGGG)2 and (TTAGGG)4 can all form quadruplex in vitro and exist mainly as parallel quadruplex without metal ions. Both K+ and Na+ can stabilize the tetrameric structure and facilitate the forming of anti-parallel conformation. Furthermore, the conformations of quadruplex can also be affected by sequence length, the nature and concentration of metal ions.

  12. Molecular dynamics of DNA quadruplex molecules containing inosine, 6-thioguanine and 6-thiopurine.

    Science.gov (United States)

    Stefl, R; Spacková, N; Berger, I; Koca, J; Sponer, J

    2001-01-01

    The ability of the four-stranded guanine (G)-DNA motif to incorporate nonstandard guanine analogue bases 6-oxopurine (inosine, I), 6-thioguanine (tG), and 6-thiopurine (tI) has been investigated using large-scale molecular dynamics simulations. The simulations suggest that a G-DNA stem can incorporate inosines without any marked effect on its structure and dynamics. The all-inosine quadruplex stem d(IIII)(4) shows identical dynamical properties as d(GGGG)(4) on the nanosecond time scale, with both molecular assemblies being stabilized by monovalent cations residing in the channel of the stem. However, simulations carried out in the absence of these cations show dramatic differences in the behavior of d(GGGG)(4) and d(IIII)(4). Whereas vacant d(GGGG)(4) shows large fluctuations but does not disintegrate, vacant d(IIII)(4) is completely disrupted within the first nanosecond. This is a consequence of the lack of the H-bonds involving the N2 amino group that is not present in inosine. This indicates that formation of the inosine quadruplex could involve entirely different intermediate structures than formation of the guanosine quadruplex, and early association of cations in this process appears to be inevitable. In the simulations, the incorporation of 6-thioguanine and 6-thiopurine sharply destabilizes four-stranded G-DNA structures, in close agreement with experimental data. The main reason is the size of the thiogroup leading to considerable steric conflicts and expelling the cations out of the channel of the quadruplex stem. The G-DNA stem can accommodate a single thioguanine base with minor perturbations. Incorporation of a thioguanine quartet layer is associated with a large destabilization of the G-DNA stem whereas the all-thioguanine quadruplex immediately collapses.

  13. Effects of Sequence Partitioning on Compression Rate

    CERN Document Server

    Alagoz, B Baykant

    2010-01-01

    In the paper, a theoretical work is done for investigating effects of splitting data sequence into packs of data set. We proved that a partitioning of data sequence is possible to find such that the entropy rate at each subsequence is lower than entropy rate of the source. Effects of sequence partitioning on overall compression rate are argued on the bases of partitioning statistics, and then, an optimization problem for an optimal partition is defined to improve overall compression rate of a sequence.

  14. Identification of the DNA-Binding Domains of Human Replication Protein A That Recognize G-Quadruplex DNA

    Directory of Open Access Journals (Sweden)

    Aishwarya Prakash

    2011-01-01

    Full Text Available Replication protein A (RPA, a key player in DNA metabolism, has 6 single-stranded DNA-(ssDNA- binding domains (DBDs A-F. SELEX experiments with the DBDs-C, -D, and -E retrieve a 20-nt G-quadruplex forming sequence. Binding studies show that RPA-DE binds preferentially to the G-quadruplex DNA, a unique preference not observed with other RPA constructs. Circular dichroism experiments show that RPA-CDE-core can unfold the G-quadruplex while RPA-DE stabilizes it. Binding studies show that RPA-C binds pyrimidine- and purine-rich sequences similarly. This difference between RPA-C and RPA-DE binding was also indicated by the inability of RPA-CDE-core to unfold an oligonucleotide containing a TC-region 5′ to the G-quadruplex. Molecular modeling studies of RPA-DE and telomere-binding proteins Pot1 and Stn1 reveal structural similarities between the proteins and illuminate potential DNA-binding sites for RPA-DE and Stn1. These data indicate that DBDs of RPA have different ssDNA recognition properties.

  15. Enantioselective targeting left-handed Z-G-quadruplex.

    Science.gov (United States)

    Zhao, Andong; Zhao, Chuanqi; Ren, Jinsong; Qu, Xiaogang

    2016-01-25

    Herein, we report the first example where an M-enantiomer of a chiral metal complex can selectively stabilize a left-handed G-quadruplex, but its P-enantiomer cannot. The interactions between the chiral metal complexes and the left-handed G-quadruplex were evaluated by UV melting, circular dichroism, isothermal titration calorimetry, gel electrophoresis and NMR titrations.

  16. Design of a fluorescent DNA IMPLICATION logic gate and detection of Ag+ and cysteine with triphenylmethane dye/G-quadruplex complexes.

    Science.gov (United States)

    Guo, Jun-Hong; Kong, De-Ming; Shen, Han-Xi

    2010-10-15

    This paper describes the construction of a DNA IMPLICATION logic gate based on triphenylmethane (TPM) dye/G-quadruplex complexes, using Ag+ and cysteine (Cys) as the two inputs, and fluorescence intensity of the TPM dye as the output signal. Free triphenylmethane (TPM) dyes emit inherently low fluorescence signal, the formation of TPM dye/G-quadruplex complexes yielded greatly enhanced fluorescence signals from the dye, and the output signal of the gate was 1. The addition of Cys had no effect on the fluorescence signal, again yielding an output of 1. However, the addition of Ag+ instead of Cys greatly disrupted the G-quadruplex structure, causing a decrease in the fluorescence of the dye, and yielding an output signal of 0. The addition of Cys into the Ag+-quenched fluorescence system led to the release of Ag+ from G-quadruplex-forming DNAs, resulting in the reformation of G-quadruplex structures and the recovery of TMP dye fluorescence, the output signal of 1 was obtained again. Compared with previously published DNA logic gates, the gate operation described here was rapid and reversible, with a reliable, nondestructive readout and excellent digital behavior. In addition, the modulation of TPM dye/G-quadruplex complex fluorescence by Ag+ and Cys could be used to develop a simple, fast, label-free and highly specific homogenous sensing methods for Ag+ and Cys. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer

    Science.gov (United States)

    Wolfe, Andrew L.; Singh, Kamini; Zhong, Yi; Drewe, Philipp; Rajasekhar, Vinagolu K.; Sanghvi, Viraj R.; Mavrakis, Konstantinos J.; Jiang, Man; Roderick, Justine E.; van der Meulen, Joni; Schatz, Jonathan H.; Rodrigo, Christina M.; Zhao, Chunying; Rondou, Pieter; de Stanchina, Elisa; Teruya-Feldstein, Julie; Kelliher, Michelle A.; Speleman, Frank; Porco, John A.; Pelletier, Jerry; Rätsch, Gunnar; Wendel, Hans-Guido

    2014-09-01

    The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

  18. Structural Dynamics of Human Telomeric G-Quadruplex Loops Studied by Molecular Dynamics Simulations

    Science.gov (United States)

    Zhu, Hong; Xiao, Shiyan; Liang, Haojun

    2013-01-01

    Loops which are linkers connecting G-strands and supporting the G-tetrad core in G-quadruplex are important for biological roles of G-quadruplexes. TTA loop is a common sequence which mainly resides in human telomeric DNA (hTel) G-quadruplex. A series of molecular dynamics (MD) simulations were carried out to investigate the structural dynamics of TTA loops. We found that (1) the TA base pair formed in TTA loops are very stable, the occupied of all hydrogen bonds are more than 0.95. (2) The TA base pair makes the adjacent G-quartet more stable than others. (3) For the edgewise loop and the diagonal loop, most loop bases are stacking with others, only few bases have considerable freedom. (4) The stabilities of these stacking structures are distinct. Part of the loops, especially TA base pairs, and bases stacking with the G-quartet, maintain certain stable conformations in the simulation, but other parts, like TT and TA stacking structures, are not stable enough. For the first time, spontaneous conformational switches of TTA edgewise loops were observed in our long time MD simulations. (5) For double chain reversal loop, it is really hard to maintain a stable conformation in the long time simulation under present force fields (parm99 and parmbsc0), as it has multiple conformations with similar free energies. PMID:23951152

  19. Structural dynamics of human telomeric G-quadruplex loops studied by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Hong Zhu

    Full Text Available Loops which are linkers connecting G-strands and supporting the G-tetrad core in G-quadruplex are important for biological roles of G-quadruplexes. TTA loop is a common sequence which mainly resides in human telomeric DNA (hTel G-quadruplex. A series of molecular dynamics (MD simulations were carried out to investigate the structural dynamics of TTA loops. We found that (1 the TA base pair formed in TTA loops are very stable, the occupied of all hydrogen bonds are more than 0.95. (2 The TA base pair makes the adjacent G-quartet more stable than others. (3 For the edgewise loop and the diagonal loop, most loop bases are stacking with others, only few bases have considerable freedom. (4 The stabilities of these stacking structures are distinct. Part of the loops, especially TA base pairs, and bases stacking with the G-quartet, maintain certain stable conformations in the simulation, but other parts, like TT and TA stacking structures, are not stable enough. For the first time, spontaneous conformational switches of TTA edgewise loops were observed in our long time MD simulations. (5 For double chain reversal loop, it is really hard to maintain a stable conformation in the long time simulation under present force fields (parm99 and parmbsc0, as it has multiple conformations with similar free energies.

  20. A mRNA-Responsive G-Quadruplex-Based Drug Release System

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2015-04-01

    Full Text Available G-quadruplex-based drug delivery carriers (GDDCs were designed to capture and release a telomerase inhibitor in response to a target mRNA. Hybridization between a loop on the GDDC structure and the mRNA should cause the G-quadruplex structure of the GDDC to unfold and release the bound inhibitor, anionic copper(II phthalocyanine (CuAPC. As a proof of concept, GDDCs were designed with a 10-30-mer loop, which can hybridize with a target sequence in epidermal growth factor receptor (EGFR mRNA. Structural analysis using circular dichroism (CD spectroscopy showed that the GDDCs form a (3 + 1 type G-quadruplex structure in 100 mM KCl and 10 mM MgCl2 in the absence of the target RNA. Visible absorbance titration experiments showed that the GDDCs bind to CuAPC with Ka values of 1.5 × 105 to 5.9 × 105 M−1 (Kd values of 6.7 to 1.7 μM at 25 °C, depending on the loop length. Fluorescence titration further showed that the G-quadruplex structure unfolds upon binding to the target RNA with Ka values above 1.0 × 108 M−1 (Kd values below 0.01 μM at 25 °C. These results suggest the carrier can sense and bind to the target RNA, which should result in release of the bound drug. Finally, visible absorbance titration experiments demonstrated that the GDDC release CuAPC in response to the target RNA.

  1. Classification of g-quadruplex DNA on the basis of the quadruplex twist angle and planarity of g-quartets.

    Science.gov (United States)

    Reshetnikov, R V; Kopylov, A M; Golovin, A V

    2010-10-01

    The present work is devoted to the analysis of the G-quadruplex DNA structure using the bioinformatics method. The interest towards quadruplex DNAs is determined by their involvement in the functioning of telomeres and onco-promoters as well as by the possibility to create on their basis aptamers and nanostructures. Here, we present an algorithm for a general analysis of the polymorphism of the G-quadruplex structure from the data bank PDB using original parameters. 74 structures were grouped according to the following parameters: the number of DNA strands, the number of G-quartets, and the location and orientation of the connecting loops. Two quantitative parameters were used to describe the quadruplex structure: the twist angle between two adjacent quartets (analogous to that for the complementary pair in the duplex DNA) and the quartet planarity (an original parameter). The distribution patterns of these values are specific for each group of quadruplex structures and are dependent upon the type of connecting loops used (diagonal, lateral or propeller). The tetramolecular loopless parallel quadruplex was used as a comparison template. The lateral loops introduce the strongest distortion into the structure of quadruplexes: the values of the twist angles are the lowest and are not typical for the other quadruplex groups. The loops of the diagonal type introduce much weaker deformation into quadruplexes; the structures with propeller loops are characterized by the optimum geometry of G-quartets. Hence, the correlation between the twist angle and the tension in the structure of quadruplex DNA is revealed.

  2. cis sequence effects on gene expression

    Directory of Open Access Journals (Sweden)

    Jacobs Kevin

    2007-08-01

    Full Text Available Abstract Background Sequence and transcriptional variability within and between individuals are typically studied independently. The joint analysis of sequence and gene expression variation (genetical genomics provides insight into the role of linked sequence variation in the regulation of gene expression. We investigated the role of sequence variation in cis on gene expression (cis sequence effects in a group of genes commonly studied in cancer research in lymphoblastoid cell lines. We estimated the proportion of genes exhibiting cis sequence effects and the proportion of gene expression variation explained by cis sequence effects using three different analytical approaches, and compared our results to the literature. Results We generated gene expression profiling data at N = 697 candidate genes from N = 30 lymphoblastoid cell lines for this study and used available candidate gene resequencing data at N = 552 candidate genes to identify N = 30 candidate genes with sufficient variance in both datasets for the investigation of cis sequence effects. We used two additive models and the haplotype phylogeny scanning approach of Templeton (Tree Scanning to evaluate association between individual SNPs, all SNPs at a gene, and diplotypes, with log-transformed gene expression. SNPs and diplotypes at eight candidate genes exhibited statistically significant (p cis sequence effects in our study, respectively. Conclusion Based on analysis of our results and the extant literature, one in four genes exhibits significant cis sequence effects, and for these genes, about 30% of gene expression variation is accounted for by cis sequence variation. Despite diverse experimental approaches, the presence or absence of significant cis sequence effects is largely supported by previously published studies.

  3. Molecular Dynamics Simulation Study of Parallel Telomeric DNA Quadruplexes at Different Ionic Strengths: Evaluation of Water and Ion Models.

    Science.gov (United States)

    Rebič, Matúš; Laaksonen, Aatto; Šponer, Jiří; Uličný, Jozef; Mocci, Francesca

    2016-08-04

    Most molecular dynamics (MD) simulations of DNA quadruplexes have been performed under minimal salt conditions using the Åqvist potential parameters for the cation with the TIP3P water model. Recently, this combination of parameters has been reported to be problematic for the stability of quadruplex DNA, especially caused by the ion interactions inside or near the quadruplex channel. Here, we verify how the choice of ion parameters and water model can affect the quadruplex structural stability and the interactions with the ions outside the channel. We have performed a series of MD simulations of the human full-parallel telomeric quadruplex by neutralizing its negative charge with K(+) ions. Three combinations of different cation potential parameters and water models have been used: (a) Åqvist ion parameters, TIP3P water model; (b) Joung and Cheatham ion parameters, TIP3P water model; and (c) Joung and Cheatham ion parameters, TIP4Pew water model. For the combinations (b) and (c), the effect of the ionic strength has been evaluated by adding increasing amounts of KCl salt (50, 100, and 200 mM). Two independent simulations using the Åqvist parameters with the TIP3P model show that this combination is clearly less suited for the studied quadruplex with K(+) as counterions. In both simulations, one ion escapes from the channel, followed by significant deformation of the structure, leading to deviating conformation compared to that in the reference crystallographic data. For the other combinations of ion and water potentials, no tendency is observed for the channel ions to escape from the quadruplex channel. In addition, the internal mobility of the three loops, torsion angles, and counterion affinity have been investigated at varied salt concentrations. In summary, the selection of ion and water models is crucial as it can affect both the structure and dynamics as well as the interactions of the quadruplex with its counterions. The results obtained with the TIP4Pew

  4. A smart tailor-made G-clip reporter for sensitive detection of G-triplet-containing sequences.

    Science.gov (United States)

    Cai, Liang-Yuan; Nie, Ji; Zhang, Yi-Wei; Zhang, Fang-Ting; Zhou, Ying-Lin; Zhang, Xin-Xiang

    2015-05-21

    Taking advantage of the intrinsic characteristics of G-triplet-containing sequences, a pioneering tailor-made clip-like reporter containing three-fourths of a G-quadruplex is established. The reporter can clip the G triplet in the target sequence through a recognition process to form a complete G-quadruplex structure.

  5. Formation and stabilization of the telomeric antiparallel G-quadruplex and inhibition of telomerase by novel benzothioxanthene derivatives with anti-tumor activity

    Science.gov (United States)

    Zhang, Wen; Chen, Min; Ling Wu, Yan; Tanaka, Yoshimasa; Juan Ji, Yan; Lin Zhang, Su; He Wei, Chuan; Xu, Yan

    2015-09-01

    G-quadruplexes formed in telomeric DNA sequences at human chromosome ends can be a novel target for the development of therapeutics for the treatment of cancer patients. Herein, we examined the ability of six novel benzothioxanthene derivatives S1-S6 to induce the formation of and stabilize an antiparallel G-quadruplex by EMSA, UV-melting and CD techniques and the influence of S1-S6 on A549 and SGC7901 cells through real-time cell analysis, wound healing, trap assay methods. Results show that six compounds could differentially induce 26 nt G-rich oligonucleotides to form the G-quadruplex with high selectivity vs C-rich DNA, mutated DNA and double-stranded DNA, stabilize it with high affinity, promote apoptosis and inhibit mobility and telomerase activity of A549 cells and SGC7901 cells. Especially, S1, S3, S4 displayed stronger abilities, of which S3 was the most optimal with the maximum ΔTm value being up to 29.8 °C for G-quadruplex, the minimum IC50 value being 0.53 μM and the maximum cell inhibitory rate being up to 97.2%. This study suggests that this type of compounds that induce the formation of and stabilize the telomeric antiparallel G-quadruplex, and consequently inhibit telomerase activity, leading to cell apoptosis, can be screened for the discovery of novel antitumor therapeutics.

  6. Discovery of a Family of Genomic Sequences Which Interact Specifically with the c-MYC Promoter to Regulate c-MYC Expression

    Science.gov (United States)

    Thomas, Shelia D.; Rouchka, Eric C.; Miller, Donald M.

    2016-01-01

    G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common

  7. Toward the design of new DNA G-quadruplex ligands through rational analysis of polymorphism and binding data.

    Science.gov (United States)

    Artese, Anna; Costa, Giosuè; Distinto, Simona; Moraca, Federica; Ortuso, Francesco; Parrotta, Lucia; Alcaro, Stefano

    2013-10-01

    Human telomeres play a key role in protecting chromosomal ends from fusion events; they are composed of d(TTAGGG) repeats, ranging in size from 3 to 15 kb. They form G-quadruplex DNA structures, stabilized by G-quartets in the presence of cations, and are involved in several biological processes. In particular, a telomere maintenance mechanism is provided by a specialized enzyme called telomerase, a reverse transcriptase able to add multiple copies of the 5'-GGTTAG-3' motif to the end of the G-strand of the telomere and which is over-expressed in the majority of cancer cells. The central cation has a crucial role in maintaining the stability of the structure. Based on its nature, it can be associated with different topological telomeric quadruplexes, which depend also on the orientation of the DNA strands and the syn/anti conformation of the guanines. Such a polymorphism, confirmed by the different structures deposited in the Protein Data Bank (PDB), prompted us to apply a computational protocol in order to investigate the conformational properties of a set of known G-quadruplex ligands and their molecular recognition against six different experimental models of the human telomeric sequence d[AG3(T2AG3)3]. The average AutoDock correlation between theoretical and experimental data yielded an r2 value equal to 0.882 among all the studied models. Such a result was always improved with respect to those of the single folds, with the exception of the parallel structure (r2 equal to 0.886), thus suggesting a key role of this G4 conformation in the stacking interaction network. Among the studied binders, a trisubstituted acridine and a dibenzophenanthroline derivative were well recognized by the parallel and the mixed G-quadruplex structures, allowing the identification of specific key contacts with DNA and the further design of more potent or target specific G-quadruplex ligands.

  8. Structure of a left-handed DNA G-quadruplex.

    Science.gov (United States)

    Chung, Wan Jun; Heddi, Brahim; Schmitt, Emmanuelle; Lim, Kah Wai; Mechulam, Yves; Phan, Anh Tuân

    2015-03-03

    Aside from the well-known double helix, DNA can also adopt an alternative four-stranded structure known as G-quadruplex. Implications of such a structure in cellular processes, as well as its therapeutic and diagnostic applications, have been reported. The G-quadruplex structure is highly polymorphic, but so far, only right-handed helical forms have been observed. Here we present the NMR solution and X-ray crystal structures of a left-handed DNA G-quadruplex. The structure displays unprecedented features that can be exploited as unique recognition elements.

  9. Elevated levels of G-quadruplex formation in human stomach and liver cancer tissues.

    Science.gov (United States)

    Biffi, Giulia; Tannahill, David; Miller, Jodi; Howat, William J; Balasubramanian, Shankar

    2014-01-01

    Four-stranded G-quadruplex DNA secondary structures have recently been visualized in the nuclei of human cultured cells. Here, we show that BG4, a G-quadruplex-specific antibody, can be used to stain DNA G-quadruplex structures in patient-derived tissues using immunohistochemistry. We observe a significantly elevated number of G-quadruplex-positive nuclei in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Our results suggest that G-quadruplex formation can be detected and measured in patient-derived material and that elevated G-quadruplex formation may be a characteristic of some cancers.

  10. Discovery of the first dual G-triplex/G-quadruplex stabilizing compound: a new opportunity in the targeting of G-rich DNA structures?

    Science.gov (United States)

    Amato, Jussara; Pagano, Alessia; Cosconati, Sandro; Amendola, Giorgio; Fotticchia, Iolanda; Iaccarino, Nunzia; Marinello, Jessica; De Magis, Alessio; Capranico, Giovanni; Novellino, Ettore; Pagano, Bruno; Randazzo, Antonio

    2017-05-01

    Guanine-rich DNA motifs can form non-canonical structures known as G-quadruplexes, whose role in tumorigenic processes makes them attractive drug-target candidates for cancer therapy. Recent studies revealed that the folding and unfolding pathways of G-quadruplexes proceed through a quite stable intermediate named G-triplex. Virtual screening was employed to identify a small set of putative G-triplex ligands. The G-triplex stabilizing properties of these compounds were analyzed by CD melting assay. DSC, non-denaturing gel electrophoresis, NMR and molecular modeling studies were performed to investigate the interaction between the selected compound 1 and G-rich DNA structures. Cytotoxic activity of 1 was evaluated by MTT cell proliferation assay. The experiments led to the identification of a promising hit that was shown to bind preferentially to G-triplex and parallel-stranded G-quadruplexes over duplex and antiparallel G-quadruplexes. Molecular modeling results suggested a partial end-stacking of 1 to the external G-triad/G-tetrads as a binding mode. Biological assays showed that 1 is endowed with cytotoxic effect on human osteosarcoma cells. A tandem application of virtual screening along with the experimental investigation was employed to discover a G-triplex-targeting ligand. Experiments revealed that the selected compound actually acts as a dual G-triplex/G-quadruplex stabilizer, thus stimulating further studies aimed at its optimization. The discovery of molecules able to bind and stabilize G-triplex structures is highly appealing, but their transient state makes challenging their recognition. These findings suggest that the identification of ligands with dual G-triplex/G-quadruplex stabilizing properties may represent a new route for the design of anticancer agents targeting the G-rich DNA structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B

  11. Identifying the impact of G-quadruplexes on Affymetrix 3' arrays using cloud computing.

    Science.gov (United States)

    Memon, Farhat N; Owen, Anne M; Sanchez-Graillet, Olivia; Upton, Graham J G; Harrison, Andrew P

    2010-01-15

    A tetramer quadruplex structure is formed by four parallel strands of DNA/ RNA containing runs of guanine. These quadruplexes are able to form because guanine can Hoogsteen hydrogen bond to other guanines, and a tetrad of guanines can form a stable arrangement. Recently we have discovered that probes on Affymetrix GeneChips that contain runs of guanine do not measure gene expression reliably. We associate this finding with the likelihood that quadruplexes are forming on the surface of GeneChips. In order to cope with the rapidly expanding size of GeneChip array datasets in the public domain, we are exploring the use of cloud computing to replicate our experiments on 3' arrays to look at the effect of the location of G-spots (runs of guanines). Cloud computing is a recently introduced high-performance solution that takes advantage of the computational infrastructure of large organisations such as Amazon and Google. We expect that cloud computing will become widely adopted because it enables bioinformaticians to avoid capital expenditure on expensive computing resources and to only pay a cloud computing provider for what is used. Moreover, as well as financial efficiency, cloud computing is an ecologically-friendly technology, it enables efficient data-sharing and we expect it to be faster for development purposes. Here we propose the advantageous use of cloud computing to perform a large data-mining analysis of public domain 3' arrays.

  12. Synthesis and G-Quadruplex-Binding Properties of Defined Acridine Oligomers

    Directory of Open Access Journals (Sweden)

    Rubén Ferreira

    2010-01-01

    Full Text Available The synthesis of oligomers containing two or three acridine units linked through 2-aminoethylglycine using solid-phase methodology is described. Subsequent studies on cell viability showed that these compounds are not cytotoxic. Binding to several DNA structures was studied by competitive dialysis, which showed a clear affinity for DNA sequences that form G-quadruplexes and parallel triplexes. The fluorescence spectra of acridine oligomers were affected strongly upon binding to DNA. These spectral changes were used to calculate the binding constants (K. Log K were found to be in the order of 4–6.

  13. Cyclic dinucleotide detection with riboswitch-G-quadruplex hybrid.

    Science.gov (United States)

    Tsuji, Genichiro; Sintim, Herman O

    2016-03-01

    A cyclic dinucleotide riboswitch has been fused with a G-quadruplex motif to produce a conditional riboswitch-peroxidase-mimicking sensor that oxidizes both colorimetric and fluorogenic substrates in the presence of c-di-GMP. We find that signal-to-noise ratio could be improved by using a two-, not three-, floor split G-quadruplex for this conditional peroxidase-mimicking riboswitch.

  14. A label-free G-quadruplex-based luminescent switch-on assay for the selective detection of histidine.

    Science.gov (United States)

    He, Hong-Zhang; Wang, Modi; Chan, Daniel Shiu-Hin; Leung, Chung-Hang; Qiu, Jian-Wen; Ma, Dik-Lung

    2013-12-15

    A label-free G-quadruplex-based luminescent switch-on assay has been developed for the selective detection of micromolar histidine in aqueous solution. In this study, an iridium(III) complex was employed as a G-quadruplex-specific luminescent probe while a guanine-rich oligonucleotide (Pu27, 5'-TG4AG3TG4AG3TG4A2G2-3')/cupric ion (Cu(2+)) ensemble was employed as a recognition unit for histidine. The initial luminescence of the iridium(III) complex in the presence of G-quadruplex DNA is effectively quenched by Cu(2+) ions due to the Cu(2+)-mediated unfolding of the G-quadruplex motif. The addition of histidine sequesters Cu(2+) ions from the ensemble, thereby restoring the luminescence of the system. The assay could detect down to 1 μM of histidine in aqueous media, and also exhibited good selectivity for histidine over other amino acids with the use of the cysteine, masking agent N-ethylmaleimide. Furthermore, the application of the assay for the detection of histidine in diluted urine samples was demonstrated.

  15. Rotation of Guanine Amino Groups in G-Quadruplexes: A Probe for Local Structure and Ligand Binding.

    Science.gov (United States)

    Adrian, Michael; Winnerdy, Fernaldo Richtia; Heddi, Brahim; Phan, Anh Tuân

    2017-08-22

    Nucleic acids are dynamic molecules whose functions may depend on their conformational fluctuations and local motions. In particular, amino groups are dynamic components of nucleic acids that participate in the formation of various secondary structures such as G-quadruplexes. Here, we present a cost-efficient NMR method to quantify the rotational dynamics of guanine amino groups in G-quadruplex nucleic acids. An isolated spectrum of amino protons from a specific tetrad-bound guanine can be extracted from the nuclear Overhauser effect spectroscopy spectrum based on the close proximity between the intra-residue imino and amino protons. We apply the method in different structural contexts of G-quadruplexes and their complexes. Our results highlight the role of stacking and hydrogen-bond interactions in restraining amino-group rotation. The measurement of the rotation rate of individual amino groups could give insight into the dynamic processes occurring at specific locations within G-quadruplex nucleic acids, providing valuable probes for local structure, dynamics, and ligand binding. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Synthesis and evaluation of 7-substituted-5,6-dihydrobenzo[c]acridine derivatives as new c-KIT promoter G-quadruplex binding ligands.

    Science.gov (United States)

    Guo, Qian-Liang; Su, Hua-Fei; Wang, Ning; Liao, Sheng-Rong; Lu, Yu-Ting; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Huang, Zhi-Shu

    2017-04-21

    It has been shown that treatment of cancer cells with c-KIT G-quadruplex binding ligands can reduce their c-KIT expression levels thus inhibiting cell proliferation and inducing cell apoptosis. Herein, a series of new 7-substituted-5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized. Subsequent biophysical evaluation demonstrated that the derivatives could effectively bind to and stabilize c-KIT G-quadruplex with good selectivity against duplex DNA. It was found that 12-N-methylated derivatives with a positive charge introduced at 12-position of 5,6-dihydrobenzo[c]acridine ring had similar binding affinity but lower stabilizing ability to c-KIT G-quadruplex DNA, compared with those of nonmethylated derivatives. Further molecular modeling studies showed possible binding modes of G-quadruplex with the ligands. RT-PCR assay and Western blot showed that compound 2b suppressed transcription and translation of c-KIT gene in K562 cells, which was consistent with the property of an effective G-quadruplex binding ligand targeting c-KIT oncogene promoter. Further biological evaluation showed that compound 2b could induce apoptosis through activation of the caspase-3 cascade pathway.

  17. Sequence selective naked-eye detection of DNA harnessing extension of oligonucleotide-modified nucleotides.

    Science.gov (United States)

    Verga, Daniela; Welter, Moritz; Marx, Andreas

    2016-02-01

    DNA polymerases can efficiently and sequence selectively incorporate oligonucleotide (ODN)-modified nucleotides and the incorporated oligonucleotide strand can be employed as primer in rolling circle amplification (RCA). The effective amplification of the DNA primer by Φ29 DNA polymerase allows the sequence-selective hybridisation of the amplified strand with a G-quadruplex DNA sequence that has horse radish peroxidase-like activity. Based on these findings we develop a system that allows DNA detection with single-base resolution by naked eye.

  18. Effect of rubidium and cesium ions on the dimeric quaduplex formed by the Oxytricha nova telomeric repeat oligonucleotide d(GGGGTTTTGGGG).

    Science.gov (United States)

    Marincola, Flaminia Cesare; Virno, Ada; Randazzo, Antonio; Lai, Adolfo

    2007-01-01

    The DNA sequence d(GGGGTTTTGGGG) consists of 1.5 units of the repeat in telomeres of Oxytricha nova. It has been shown by NMR and x-ray crystallographic analysis that it is capable to form a dimeric quadruplex structure and that a variety of cations, namely K(+), Na(+), and NH(4)(+), are able to interact with this complex with different affinity, leading to complexes characterized by different local conformations. Thus, in order to improve the knowledge of this kind of molecule, and in particular to provide further insight into the role of monovalent cations in the G-quadruplex folding and conformation, we have investigated by (1)H-NMR the effect of the addition of Rb(+) and Cs(+) to the quadruplex formed by the oligonucleotide d(GGGGTTTTGGGG).

  19. General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

    Science.gov (United States)

    Chang, Tianjun; Qi, Cui; Meng, Jie; Zhang, Nan; Bing, Tao; Yang, Xianda; Cao, Zehui; Shangguan, Dihua

    2013-01-01

    G-quadruplexes (G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs) are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes) have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.

  20. General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

    Directory of Open Access Journals (Sweden)

    Tianjun Chang

    Full Text Available G-quadruplexes (G4s are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.

  1. Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G-Quadruplexes.

    Science.gov (United States)

    Dang, Dung Thanh; Phan, Anh Tuân

    2016-01-01

    We have developed fluorescent protein probes specific for parallel G-quadruplexes by attaching cyan fluorescent protein to the G-quadruplex-binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G-quadruplexes was characterized. The selective recognition and discrimination of G-quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.

  2. Targeting human telomeric G-quadruplex DNA and inhibition of telomerase activity with [(dmb2Ru(obipRu(dmb2](4+.

    Directory of Open Access Journals (Sweden)

    Shuo Shi

    Full Text Available Inhibition of telomerase by inducing/stabilizing G-quadruplex formation is a promising strategy to design new anticancer drugs. We synthesized and characterized a new dinuclear complex [(dmb2Ru(obipRu(dmb2](4+ (dmb = 4,4'-dimethyl-2,2'-bipyridine, obip = (2-(2-pyridylimidazo[4,5-f][1,10]phenanthroline with high affinity for both antiparallel and mixed parallel / antiparallel G-quadruplex DNA. This complex can promote the formation and stabilize G-quadruplex DNA. Dialysis and TRAP experiments indicated that [(dmb2Ru(obipRu(dmb2](4+ acted as an excellent telomerase inhibitor due to its obvious selectivity for G-quadruplex DNA rather than double stranded DNA. In vitro co-culture experiments implied that [(dmb2Ru(obipRu(dmb2](4+ inhibited telomerase activity and hindered cancer cell proliferation without side effects to normal fibroblast cells. TUNEL assay indicated that inhibition of telomerase activity induced DNA cleavage further apoptosis in cancer cells. Therefore, Ru(II complex represents an exciting opportunity for anticancer drug design by specifically targeting cancer cell G-quadruplexes DNA.

  3. Programing the formation of DNA and PNA quadruplexes by pi-pi-stacking interactions.

    Science.gov (United States)

    Saha, Sourav; Cai, Jianfeng; Eiler, Daniel; Hamilton, Andrew D

    2010-03-14

    Guanine (G) rich G(4)T(4)G(4) DNA and homologous PNA strands tend to form antiparallel dimeric quadruplexes. In contrast, the same DNA strands carrying planar aromatic 5'-residues preferentially form parallel DNA quadruplex. Conformation and composition of the DNA quadruplexes can be programed by pi-pi-stacking interaction exerted by the 5'-residues.

  4. Studies on non-covalent interaction of coumarin attached pyrimidine and 1-methyl indole 1,2,3 triazole analogues with intermolecular telomeric G-quadruplex DNA using ESI-MS and spectroscopy.

    Science.gov (United States)

    Raju, G; Srinivas, R; Reddy, M Damoder; Reddy, Ch Raji; Nagesh, N

    2014-01-01

    In the present study, electrospray ionization mass spectrometry (ESI-MS) and spectroscopy have been used to evaluate the non-covalent interaction, stoichiometry, and selectivity of two synthetic coumarin-attached nucleoside and non-nucleoside 1,2,3-triazoles, namely, (1-(5-(hydroxymethyl)-4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-furan-2-yl)5-methyl pyrimidine-2,4(1H,3H)-dione (Tr1) and 4-((1-((-1-methyl-1H-indol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (Tr2) with two different human telomeric intermolecular G-quadruplex DNA structures formed by d(T2AG3) and d(T2AG3)2 sequences. ESI-MS studies indicate that Tr1 specifically interacts with four-stranded intermolecular parallel quadruplex complex, whereas Tr2 interacts with two hairpin as well as four-stranded intermolecular parallel quadruplex complexes. UV-Visible spectroscopic studies suggest that Tr1 and Tr2 interact with G-quadruplex structure and unwind them. Job plots show that stoichiometry of ligand:quadruplex DNA is 1:1. Circular dichroism (CD) studies of G-quadruplex DNA and Tr1/Tr2 ligands manifest that they unfold DNA on interaction. Fluorescence studies demonstrate that ligand molecules intercalate between the two stacks of quadruplex DNA and non-radiative energy transfer occurs between the excited ligand molecules (donor) and quadruplex DNA (acceptor), resulting in enhancement of fluorescence emission intensity. Thus, these studies suggest that nucleoside and non-nucleoside ligands efficiently interact with d(T2AG3) and d(T2AG3)2 G-quadruplex DNA but the interaction is not alike with all kinds of quadruplex DNA, this is probably due to the variation in the pharmacophores and structure of the ligand molecules.

  5. G Quadruplex in Plants: A Ubiquitous Regulatory Element and Its Biological Relevance

    Directory of Open Access Journals (Sweden)

    Vikas Yadav

    2017-07-01

    Full Text Available G quadruplexes (G4 are higher-order DNA and RNA secondary structures formed by G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential G4 quadruplex sequences have been identified in G-rich eukaryotic non-telomeric and telomeric genomic regions. Upon function, G4 formation is known to involve in chromatin remodeling, gene regulation and has been associated with genomic instability, genetic diseases and cancer progression. The natural role and biological validation of G4 structures is starting to be explored, and is of particular interest for the therapeutic interventions for human diseases. However, the existence and physiological role of G4 DNA and G4 RNA in plants species have not been much investigated yet and therefore, is of great interest for the development of improved crop varieties for sustainable agriculture. In this context, several recent studies suggests that these highly diverse G4 structures in plants can be employed to regulate expression of genes involved in several pathophysiological conditions including stress response to biotic and abiotic stresses as well as DNA damage. In the current review, we summarize the recent findings regarding the emerging functional significance of G4 structures in plants and discuss their potential value in the development of improved crop varieties.

  6. Assaying the binding strength of G-quadruplex ligands using single-molecule TPM experiments.

    Science.gov (United States)

    Liu, Shih-Wei; Chu, Jen-Fei; Tsai, Cheng-Ting; Fang, Hung-Chih; Chang, Ta-Chau; Li, Hung-Wen

    2013-05-15

    G-quadruplexes are stable secondary structures formed by Hoogsteen base pairing of guanine-rich single-stranded DNA sequences in the presence of monovalent cations (Na(+) or K(+)). Folded G-quadruplex (G4) structures in human telomeres have been proposed as a potential target for cancer therapy. In this study, we used single-molecule tethered particle motion (TPM) experiments to assay the binding strength of possible G4 ligands. We found that individual single-stranded DNA molecules containing the human telomeric sequence d[AGGG(TTAGGG)3] fluctuated between the folded and the unfolded states in a 10 mM Na(+) solution at 37 °C. The durations of folded and unfolded states were single-exponentially distributed, and in return the folding and unfolding rate constants were 1.68 ± 0.01 and 1.63 ± 0.03 (s(-1)), respectively. In the presence of G4 ligands, such as TMPyP4, DODCI, BMVC, and BMVPA, the unfolding rate constant decreased appreciably. In addition, combining the Cu(2+)-induced G4 unfolding and TPM assay, we showed that BMVC and TMPyP4 are better G4 stabilizers than DODCI. The capability of monitoring the fluctuation between the folded and the unfolded state of G4 DNA in real time allows the determination of both kinetic and thermodynamic parameters in a single measurement and offers a simple way to assay binding strength under various conditions.

  7. Finding a human telomere DNA-RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: a protective structure for telomere end.

    Science.gov (United States)

    Xu, Yan; Suzuki, Yuta; Ishizuka, Takumi; Xiao, Chao-Da; Liu, Xiao; Hayashi, Tetsuya; Komiyama, Makoto

    2014-08-15

    Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA-RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA-RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA-RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA-RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA-RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.

  8. Dual recognition of the human telomeric G-quadruplex by a neomycin-anthraquinone conjugate.

    Science.gov (United States)

    Ranjan, Nihar; Davis, Erik; Xue, Liang; Arya, Dev P

    2013-06-28

    The authors report the recognition of a G-quadruplex formed by four repeat human telomeric DNA with aminosugar intercalator conjugates. The recognition of the G-quadruplex through dual binding mode ligands significantly increased the affinity of ligands for the G-quadruplex. One such example is a neomycin-anthraquinone conjugate (2) which exhibited nanomolar affinity for the quadruplex, and the affinity of (2) is nearly 1000 fold higher for the human telomeric G-quadruplex DNA than its constituent units, neomycin and anthraquinone.

  9. Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, Mads Corvinius; Larsen, Anders Foller; Abdikadir, Faisal Hussein

    2014-01-01

    G-quadruplex (G4) ligands are currently receiving considerable attention as potential anticancer therapeutics. A series of phenanthroline-2,9-bistriazoles carrying tethered positive end groups has been synthesized and evaluated as G4 stabilizers. The compounds were efficiently assembled by copper......(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in CH2Cl2 and water in the presence of a complexing agent. Characterization of the target compounds on telomeric and c-KIT G4 sequences led to the identification of guanidinium-substituted compounds as potent G4 DNA ligands with high selectivity over duplex DNA....... The diisopropylguanidium ligands exhibited high selectivity for the proto-oncogenic sequence c-KIT over the human telomeric sequence in the surface plasmon resonance (SPR) assay, whereas the compounds appeared potent on both G4 structures in the FRET melting temperature assay. The phenanthroline-2,9-bistriazole ligands...

  10. Differential scanning calorimetry to investigate G-quadruplexes structural stability.

    Science.gov (United States)

    Pagano, Bruno; Randazzo, Antonio; Fotticchia, Iolanda; Novellino, Ettore; Petraccone, Luigi; Giancola, Concetta

    2013-11-01

    Differential Scanning Calorimetry (DSC) is a straightforward methodology to characterize the energetics of thermally-induced transitions of DNA and other biological macromolecules. Therefore, DSC has been used to study the thermodynamic stability of several nucleic acids structures. G-quadruplexes are among the most important non-canonical nucleic acid architectures that are receiving great consideration. This article reports examples on the contribution of DSC to the knowledge of G-quadruplex structures. The selected case studies show the potential of this method in investigating the structure stability of G-quadruplex forming nucleic acids, and in providing information on their structural complexity. Indeed, DSC can determine thermodynamic parameters of G-quadruplex folding/unfolding processes, but it can also be useful to reveal the formation of multiple conformations or the presence of intermediate states along the unfolding pathway, and to evaluate the impact of chemical modifications on their structural stability. This article aims to show that DSC is an important complementary methodology to structural techniques, such as NMR and X-ray crystallography, in the study of G-quadruplex forming nucleic acids. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Characterization of Quadruplex DNA Structure by Circular Dichroism.

    Science.gov (United States)

    Del Villar-Guerra, Rafael; Gray, Robert D; Chaires, Jonathan B

    2017-03-02

    Circular dichroism (CD) is a phenomenon that arises from the differential absorption of left- and right-handed circularly polarized light, and may be seen with optically active molecules. CD spectroscopy provides useful spectral signatures for biological macromolecules in solution, and provides low-resolution structural information about macromolecular conformation. CD spectroscopy is particularly useful for monitoring conformational changes in macromolecules upon environmental perturbations. G-quadruplex structures show unique CD spectral signatures, and CD is an important tool for characterizing their formation and global structure. This protocol offers step-by-step methods for determining reliable and reproducible CD spectra of quadruplex structures and normalizing the spectra for presentation. CD spectra properly normalized with respect to quadruplex concentration and path length are required to facilitate accurate comparison of results among laboratories. The standard operating procedures proposed are recommended to make such comparison accurate and informative. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  12. Exponential quadruplex priming amplification for DNA-based isothermal diagnostics.

    Science.gov (United States)

    Partskhaladze, Tamar; Taylor, Adam; Lomidze, Levan; Gvarjaladze, David; Kankia, Besik

    2015-02-01

    Polymerase chain reaction (PCR) is a method of choice for molecular diagnostics. However, PCR relies on thermal cycling, which is not compatible with the goals of point-of-care diagnostics. A simple strategy to turn PCR into an isothermal method would be to use specific primers, which upon polymerase elongation can self-dissociate from the primer-binding sites. We recently demonstrated that a monomolecular DNA quadruplex, GGGTGGGTGGGTGGG, meets these requirements, which led to the development of the linear versions of quadruplex priming amplification (QPA). Here we demonstrate exponential version of isothermal QPA, which allows an unprecedented 10(10)-fold amplification of DNA signal in less than 40 min.

  13. Prospect of bioflavonoid fisetin as a quadruplex DNA ligand: a biophysical approach.

    Directory of Open Access Journals (Sweden)

    Bidisha Sengupta

    Full Text Available Quadruplex (G4 forming sequences in telomeric DNA and c-myc promoter regions of human DNA are associated with tumorogenesis. Ligands that can facilitate or stabilize the formation and increase the stabilization of G4 can prevent tumor cell proliferation and have been regarded as potential anti-cancer drugs. In the present study, steady state and time-resolved fluorescence measurements provide important structural and dynamical insights into the free and bound states of the therapeutically potent plant flavonoid fisetin (3,3',4',7-tetrahydroxyflavone in a G4 DNA matrix. The excited state intra-molecular proton transfer (ESPT of fisetin plays an important role in observing and understanding the binding of fisetin with the G4 DNA. Differential absorption spectra, thermal melting, and circular dichroism spectroscopic studies provide evidences for the formation of G4 DNA and size exclusion chromatography (SEC proves the binding and 1∶1 stoichiometry of fisetin in the DNA matrix. Comparative analysis of binding in the presence of EtBr proves that fisetin favors binding at the face of the G-quartet, mostly along the diagonal loop. Time resolved fluorescence anisotropy decay analysis indicates the increase in the restrictions in motion from the free to bound fisetin. We have also investigated the fingerprints of the binding of fisetin in the antiparallel quadruplex using Raman spectroscopy. Preliminary results indicate fisetin to be a prospective candidate as a G4 ligand.

  14. Circular Dichroism of DNA G-Quadruplexes: Combining Modeling and Spectroscopy To Unravel Complex Structures.

    Science.gov (United States)

    Gattuso, Hugo; Spinello, Angelo; Terenzi, Alessio; Assfeld, Xavier; Barone, Giampaolo; Monari, Antonio

    2016-03-31

    We report on the comparison between the computational and experimental determination of electronic circular dichroism spectra of different guanine quadruplexes obtained from human telomeric sequences. In particular the difference between parallel, antiparallel, and hybrid structures is evidenced, as well as the induction of transitions between the polymorphs depending on the solution environment. Extensive molecular dynamics simulations (MD) are used to probe the conformational space of the different quadruplexes, and subsequently state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM) techniques coupled with excitonic semiempirical Hamiltonian are used to simulate the macromolecular induced circular dichroism. The coupling of spectroscopy and molecular simulation allows an efficient one-to-one mapping between structures and optical properties, offering a way to disentangle the rich, yet complicated, quantity of information embedded in circular dichroism spectra. We show that our methodology is robust and efficient and allows us to take into account subtle conformational changes. As such, it could be used as an efficient tool to investigate structural modification upon DNA/drug interactions.

  15. Prospect of bioflavonoid fisetin as a quadruplex DNA ligand: a biophysical approach.

    Science.gov (United States)

    Sengupta, Bidisha; Pahari, Biswapathik; Blackmon, Laura; Sengupta, Pradeep K

    2013-01-01

    Quadruplex (G4) forming sequences in telomeric DNA and c-myc promoter regions of human DNA are associated with tumorogenesis. Ligands that can facilitate or stabilize the formation and increase the stabilization of G4 can prevent tumor cell proliferation and have been regarded as potential anti-cancer drugs. In the present study, steady state and time-resolved fluorescence measurements provide important structural and dynamical insights into the free and bound states of the therapeutically potent plant flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) in a G4 DNA matrix. The excited state intra-molecular proton transfer (ESPT) of fisetin plays an important role in observing and understanding the binding of fisetin with the G4 DNA. Differential absorption spectra, thermal melting, and circular dichroism spectroscopic studies provide evidences for the formation of G4 DNA and size exclusion chromatography (SEC) proves the binding and 1∶1 stoichiometry of fisetin in the DNA matrix. Comparative analysis of binding in the presence of EtBr proves that fisetin favors binding at the face of the G-quartet, mostly along the diagonal loop. Time resolved fluorescence anisotropy decay analysis indicates the increase in the restrictions in motion from the free to bound fisetin. We have also investigated the fingerprints of the binding of fisetin in the antiparallel quadruplex using Raman spectroscopy. Preliminary results indicate fisetin to be a prospective candidate as a G4 ligand.

  16. Investigating actinomycin D binding to G-quadruplex, i-motif and double-stranded DNA in 27-nt segment of c-MYC gene promoter.

    Science.gov (United States)

    Niknezhad, Zhila; Hassani, Leila; Norouzi, Davood

    2016-01-01

    c-MYC DNA is an attractive target for drug design, especially for cancer chemotherapy. Around 90% of c-MYC transcription is controlled by NHE III1, whose 27-nt purine-rich strand has the ability to form G-quadruplex structure. In this investigation, interaction of ActD with 27-nt G-rich strand (G/c-MYC) and its equimolar mixture with the complementary sequence, (GC/c-MYC) as well as related C-rich oligonucleotide (C/c-MYC) was evaluated. Molecular dynamic simulations showed that phenoxazine and lactone rings of ActD come close to the outer G-tetrad nucleotides indicating that ActD binds through end-stacking to the quadruplex DNA. RMSD and RMSF revealed that fluctuation of the quadruplex DNA increases upon interaction with the drug. The results of spectrophotometry and spectrofluorometry indicated that ActD most probably binds to the c-MYC quadruplex and duplex DNA via end-stacking and intercalation, respectively and polarity of ActD environment decreases due to the interaction. It was also found that binding of ActD to the GC-rich DNA is stronger than the two other forms of DNA. Circular dichroism results showed that the type of the three forms of DNA structures doesn't change, but their compactness alters due to their interaction with ActD. Finally, it can be concluded that ActD binds differently to double stranded DNA, quadruplex DNA and i-motif. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility.

    Science.gov (United States)

    Stefanovic, Snezana; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3'-untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3' UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson-Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site. © 2014 Stefanovic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. Platinum(II) phenanthroimidazole G-quadruplex ligand induces selective telomere shortening in A549 cancer cells.

    Science.gov (United States)

    Mancini, Johanna; Rousseau, Philippe; Castor, Katherine J; Sleiman, Hanadi F; Autexier, Chantal

    2016-02-01

    Telomere maintenance, achieved by the binding of protective shelterin capping proteins to telomeres and by either telomerase or a recombination-based alternative lengthening of telomere (ALT) mechanism, is critical for cell proliferation and survival. Extensive telomere shortening or loss of telomere integrity activates DNA damage checkpoints, leading to cell senescence or death. Although telomerase upregulation is an attractive target for anti-cancer therapy, the lag associated with telomere shortening and the potential activation of ALT pose a challenge. An alternative approach is to modify telomere interactions with binding proteins (telomere uncapping). G-quadruplex ligands stabilize structures generated from single-stranded G-rich 3'-telomere end (G-quadruplex) folding, which in principle, cannot be elongated by telomerase, thus leading to telomere shortening. Ligands can also mediate rapid anti-proliferative effects by telomere uncapping. We previously reported that the G-quadruplex ligand, phenylphenanthroimidazole ethylenediamine platinum(II) (PIP), inhibits telomerase activity in vitro[47]. In the current study, a long-term seeding assay showed that PIP significantly inhibited the seeding capacity of A549 lung cancer cells and to a lesser extent primary MRC5 fibroblast cells. Importantly, treatment with PIP caused a significant dose- and time-dependent decrease in average telomere length of A549 but not MRC5 cells. Moreover, cell cycle analysis revealed a significant increase in G1 arrest upon treatment of A549 cells, but not MRC5 cells. Both apoptosis and cellular senescence may contribute to the anti-proliferative effects of PIP. Our studies validate the development of novel and specific therapeutic ligands targeting telomeric G-quadruplex structures in cancer cells.

  19. Interactions of daidzin with intramolecular G-quadruplex.

    Science.gov (United States)

    Li, Wei; Zhang, Ming; Zhang, Jin-li; Li, Hui-qing; Zhang, Xiao-chen; Sun, Qian; Qiu, Chun-mei

    2006-09-04

    The potential interaction of daidzin, an ingredient of soy isoflavones, with human telomeric antiparallel G-quadruplex dAG(3)(T(2)AG(3))(3) was studied using ESI-MS, PAGE, CD and molecular simulation. Experimental studies indicated that daidzin molecules interacted with dAG(3)(T(2)AG(3))(3) and formed DNA-daidzin complex with the stoichiometric ratio of 1:1 and 1:2. The transition temperature of the G-quadruplex increased at higher ratio of daidzin to DNA. Under molecular crowding conditions the interactions between daidzin and the G-quadruplex become much stronger. Combining computational simulation and experimental results, it is demonstrated that the dAG(3)(T(2)AG(3))(3)/daidzin complex with a stoichiometric ratio of 1:1 is stabilized through the pi-pi conjugacy interactions and hydrogen bondings between daidzin and the bases of G-quadruplex. This work provides guidance not only on exploring the molecular anti-cancer mechanism of dietary isoflavones, but also searching small natural products as promising anticancer candidates that can inhibit telomerase activity.

  20. Single-molecule choreography between telomere proteins and G quadruplexes.

    Science.gov (United States)

    Hopfner, Karl-Peter

    2014-06-10

    Telomeric DNA binds proteins to protect chromosome ends, but it also adopts G quadruplex (GQ) structures. Two new studies by Hwang and colleagues (in this issue of Structure) and Ray and colleagues (published elsewhere) use single molecule imaging to reveal how GQs affect the binding of different telomere associated proteins. The data suggest that GQs play important roles in regulating accessibility of telomeres.

  1. Spectroscopic detection of DNA quadruplexes by vibrational circular dichroism.

    Science.gov (United States)

    Andrushchenko, Valery; Tsankov, Dimiter; Krasteva, Maria; Wieser, Helmut; Bour, Petr

    2011-09-28

    The four-stranded G-quadruplex motif is a conformation frequently adopted by guanine-rich nucleic acids that plays an important role in biology, medicine, and nanotechnology. Although vibrational spectroscopy has been widely used to investigate nucleic acid structure, association of particular spectral features with the quadruplex structure has to date been ambiguous. In this work, experimental IR absorption and vibrational circular dichroism (VCD) spectra of the model quadruplex systems d(G)(8) and deoxyguanosine-5'-monophosphate (5'-dGMP) were analyzed using molecular dynamics (MD) and quantum-chemical modeling. The experimental spectra were unambiguously assigned to the quadruplex DNA arrangement, and several IR and VCD bands related to this structural motif were determined. Involvement of MD in the modeling was essential for realistic simulation of the spectra. The VCD signal was found to be more sensitive to dynamical structural variations than the IR signal. The combination of the spectroscopic techniques with multiscale simulations provides extended information about nucleic acid conformations and their dynamics.

  2. More on the Cause-Effect Sequence

    Science.gov (United States)

    Janik, Jerzy A.

    2007-06-01

    Does every event have a cause? An answer is not simple. The notion of cause contains a particular being y acting on being x plus everything that may be called the boundary conditions. These may form necessary and suffcient conditions giving rise to a strong cause, or only necessary conditions, giving rise to a weak cause. These matters are discussed in this article with particular attention being paid to the argumentation of Thomas Aquinas known as prima via. Prima via is the analysis of a cause-effect sequence which leads (according to Thomas) to a First Cause (First Mover). It seems that the extrapolation of the cause-effect sequence to infinity is permissible from the logical point of view. But the possibility of weak causes seems to destroy the cause-effect "line". Here it is perhaps useful to "escape" to the metaphysical abstraction which looks at things sub ratione entitatis. If we ignore space and time (which is characteristic of this abstraction) we are led to believe that the IS of cause is finally unavoidable, which means that from the vantage point of this abstraction, i.e. from the point of view of IS, all causes are strong.

  3. Stabilizing G-quadruplex DNA by methylazacalix[n]pyridine through shape-complementary interaction.

    Science.gov (United States)

    Guan, Ai-Jiao; Shen, Meng-Jie; Zhang, En-Xuan; Li, Qian; Wang, Li-Xia; Xu, Li-Jin; Xiang, Jun-Feng; Tang, Ya-Lin

    2016-01-15

    It is found that G-quadruplexes have important functions in biological systems, such as gene expression. Molecules which can stabilize the G-quadruplex structure may have potential application in regulating the expression of gene. A series of methylazacalix[n]pyridine (n=4, 6, 7, 8, 9) has been tested to stabilize the intermolecular human telomeric G-quadruplex (T12 and H12), intramolecular TBA, c-kit and bcl-2 G-quadruplex by CD denaturation experiments. The results showed that only methylazacalix[6]pyridine (MACP6) can stabilize the intermolecular G-quadruplex formed from the 12bp human telomere. Further studies evidenced that the shape-complementary binding mode was what contributed to the interaction between MACP6 and T12 G-quadruplex.

  4. Simple and fast screening of G-quadruplex ligands with electrochemical detection system.

    Science.gov (United States)

    Fan, Qiongxuan; Li, Chao; Tao, Yaqin; Mao, Xiaoxia; Li, Genxi

    2016-11-01

    Small molecules that may facilitate and stabilize the formation of G-quadruplexes can be used for cancer treatments, because the G-quadruplex structure can inhibit the activity of telomerase, an enzyme over-expressed in many cancer cells. Therefore, there is considerable interest in developing a simple and high-performance method for screening small molecules binding to G-quadruplex. Here, we have designed a simple electrochemical approach to screen such ligands based on the fact that the formation and stabilization of G-quadruplex by ligand may inhibit electron transfer of redox species to electrode surface. As a proof-of-concept study, two types of classical G-quadruplex ligands, TMPyP4 and BRACO-19, are studied in this work, which demonstrates that this method is fast and robust and it may be applied to screen G-quadruplex ligands for anticancer drugs testing and design in the future.

  5. Spectral properties and thermal stability of AS1411 G-quadruplex.

    Science.gov (United States)

    Bagheri, Zeinab; Ranjbar, Bijan; Latifi, Hamid; Zibaii, Mohammad Ismail; Moghadam, Tahereh Tohidi; Azizi, Azade

    2015-01-01

    G-quadruplexes are supramolecular structures of G-rich nucleic acid, formed by non-canonical base pairing in the presence of specific environmental inducers. These structures have been vastly considered in diagnostic and therapeutic applications. However, detailed information on structure, optical properties and thermal stability of G-quadruplex potent oligonucleotides is scarce. Herein, optical properties and thermodynamic stability of AS1411 quadruplex is reported for various concentrations of potassium and lead ions. Circular dichroism showed that AS1411 ss-DNA folds into parallel conformation in the presence of metal ions and molecular crowding condition. UV-vis spectroscopy indicated formation of quadruplex and fluorescent spectroscopy revealed intercalation of PicoGreen in its structure, with enhancement of emission intensity upon increment of metal ion concentration. This investigation also proposes high-throughput and reliable analysis of AS1411 quadruplex's thermal stability by real-time PCR technique, which can be further applied for other quadruplex structures.

  6. Effects of an Additional Sequence of Color Stimuli on Visuomotor Sequence Learning

    Science.gov (United States)

    Tanaka, Kanji; Watanabe, Katsumi

    2017-01-01

    Through practice, people are able to integrate a secondary sequence (e.g., a stimulus-based sequence) into a primary sequence (e.g., a response-based sequence), but it is still controversial whether the integrated sequences lead to better learning than only the primary sequence. In the present study, we aimed to investigate the effects of a sequence that integrated space and color sequences on early and late learning phases (corresponding to effector-independent and effector-dependent learning, respectively) and how the effects differed in the integrated and primary sequences in each learning phase. In the task, the participants were required to learn a sequence of button presses using trial-and-error and to perform the sequence successfully for 20 trials (m × n task). First, in the baseline task, all participants learned a non-colored sequence, in which the response button always turned red. Then, in the learning task, the participants were assigned to two groups: a colored sequence group (i.e., space and color) or a non-colored sequence group (i.e., space). In the colored sequence, the response button turned a pre-determined color and the participants were instructed to attend to the sequences of both location and color as much as they could. The results showed that the participants who performed the colored sequence acquired the correct button presses of the sequence earlier, but showed a slower mean performance time than those who performed the non-colored sequence. Moreover, the slower performance time in the colored sequence group remained in a subsequent transfer task in which the spatial configurations of the buttons were vertically mirrored from the learning task. These results indicated that if participants explicitly attended to both the spatial response sequence and color stimulus sequence at the same time, they could develop their spatial representations of the sequence earlier (i.e., early development of the effector-independent learning), but might

  7. Structure and Stability of a Dimeric G-Quadruplex Formed by Cyclic Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Joan Casals

    2010-01-01

    containing two copies of the human telomeric repeat. In the presence of sodium, NMR data are consistent with a dimeric structure of the molecule in which two cycles self-associate forming a quadruplex with three guanine tetrads connected by edgewise loops. The two macrocycles are arranged in a parallel way, and the dimeric structure exhibits a high melting temperature. These results indicate that cyclization of the phosphodiester chain does not prevent quadruplex formation, although it affects the global topology of the quadruplex.

  8. Real time monitoring of Pb2+-induced formation of G-quadruplex DNA with LPFG sensor

    Science.gov (United States)

    Bagheri, Z.; Latifi, H.; Ranjbar, B.; Zibaii, M. I.; Behroodi, E.

    2015-09-01

    In this paper the potential of real time monitoring of AS1411 Guanine-quadruplex folding by using long period fiber grating (LPFG) biosensor was described. AS1411 ss-DNA folds into parallel bimolecular quadruplex in solution containing metal ions. While immobilized AS1411 folds, molecular crowding of sensing area will increase and cause change the refractive index (RI) around the sensor. In addition RI change is absolutely dependent on G-quadruplex factor concentration.

  9. Colorimetric and fluorescence detection of G-quadruplex nucleic acids with a coumarin-benzothiazole probe.

    Science.gov (United States)

    Yan, Jin-wu; Tian, Yi-guang; Tan, Jia-heng; Huang, Zhi-shu

    2015-11-01

    A colorimetric and red-emitting fluorescent dual probe for G-quadruplexes was devised with a conjugated coumarin-benzothiazole scaffold. Its significant and distinct changes in both color and fluorescence enable the label-free and visual detection of G-quadruplex structures. In addition, this probe gives a distinct strong emission response to the nucleoli in fixed cells imaging, which might be attributed to the interaction between the probe and rDNA G-quadruplex based on the chromatin immunoprecipitation assay. All these results suggest its promising application prospects in the G-quadruplex research field.

  10. Separation of the potential G-quadruplex ligands from the butanol extract of Zanthoxylum ailanthoides Sieb. & Zucc. by countercurrent chromatography and preparative high performance liquid chromatography.

    Science.gov (United States)

    Han, Tian; Cao, Xueli; Xu, Jing; Pei, Hairun; Zhang, Hong; Tang, Yalin

    2017-07-21

    stabilization effects on G-quadruplex by increasing G-quadruplex's Tm approximately 10°C, which may be the most potent G-quadruplex ligands in Z. ailanthoides. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

    Directory of Open Access Journals (Sweden)

    Lindsay D. Smith

    2014-10-01

    Full Text Available The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5′ end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs.

  12. Label-free logic modules and two-layer cascade based on stem-loop probes containing a G-quadruplex domain.

    Science.gov (United States)

    Guo, Yahui; Cheng, Junjie; Wang, Jine; Zhou, Xiaodong; Hu, Jiming; Pei, Renjun

    2014-09-01

    A simple, versatile, and label-free DNA computing strategy was designed by using toehold-mediated strand displacement and stem-loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade were constructed. The probes contain a G-quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light-up fluorescent signal of G-quadruplex/NMM complex was used as the output readout. The inputs are the disease-specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label-free and modular strategy might be adapted in multi-target diagnosis through DNA hybridization and aptamer-target interaction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Exploring the binding of d(GGGT)4 to the HIV-1 integrase: An approach to investigate G-quadruplex aptamer/target protein interactions.

    Science.gov (United States)

    Esposito, Veronica; Pirone, Luciano; Mayol, Luciano; Pedone, Emilia; Virgilio, Antonella; Galeone, Aldo

    2016-08-01

    The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence. This approach has allowed the identification of which part of the aptamer G-quadruplex structure is mainly involved in the interaction with the protein.

  14. Exploring the Interactions of the Dietary Plant Flavonoids Fisetin and Naringenin with G-Quadruplex and Duplex DNA, Showing Contrasting Binding Behavior: Spectroscopic and Molecular Modeling Approaches.

    Science.gov (United States)

    Bhattacharjee, Snehasish; Chakraborty, Sandipan; Sengupta, Pradeep K; Bhowmik, Sudipta

    2016-09-01

    Guanine-rich sequences have the propensity to fold into a four-stranded DNA structure known as a G-quadruplex (G4). G4 forming sequences are abundant in the promoter region of several oncogenes and become a key target for anticancer drug binding. Here we have studied the interactions of two structurally similar dietary plant flavonoids fisetin and naringenin with G4 as well as double stranded (duplex) DNA by using different spectroscopic and modeling techniques. Our study demonstrates the differential binding ability of the two flavonoids with G4 and duplex DNA. Fisetin more strongly interacts with parallel G4 structure than duplex DNA, whereas naringenin shows stronger binding affinity to duplex rather than G4 DNA. Molecular docking results also corroborate our spectroscopic results, and it was found that both of the ligands are stacked externally in the G4 DNA structure. C-ring planarity of the flavonoid structure appears to be a crucial factor for preferential G4 DNA recognition of flavonoids. The goal of this study is to explore the critical effects of small differences in the structure of closely similar chemical classes of such small molecules (flavonoids) which lead to the contrasting binding properties with the two different forms of DNA. The resulting insights may be expected to facilitate the designing of the highly selective G4 DNA binders based on flavonoid scaffolds.

  15. Binding-induced and label-free colorimetric method for protein detection based on autonomous assembly of hemin/G-quadruplex DNAzyme amplification strategy.

    Science.gov (United States)

    Wu, Hao; Zhang, Kai; Liu, Yaling; Wang, Hongyong; Wu, Jun; Zhu, Feifan; Zou, Pei

    2015-02-15

    In this work, a new binding-induced and label-free colorimetric method for protein detection has been developed on the basis of an autonomous assembly of hemin/G-quadruplex DNAzyme amplification strategy. The system consists of two proximity probes carrying two aptamer sequences as recognition elements for target, and two hairpin structures include three-fourths and one-fourth of the G-quadruplex sequences in inactive configuration as functional elements. In the presence of target protein, two proximity probes bind to the protein simultaneously, forming a stable DNA-protein complex. Then the complex triggers an autonomous cross-opening of the two functional hairpin structures, leading to the formation of numerous hemin/G-quadruplex DNAzymes. The resulting DNAzymes catalyze the oxidation of colorless 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(2-)) to the green-colored ABTS(•-) with the presence of H2O2, thus providing the amplified colorimetric detection of target. Using human α-thrombin as the protein target, this binding-induced DNAzyme amplification colorimetric method affords high sensitivity with a detection limit of 1.9 pM. Furthermore, this method might be further extended to sensitive detection of other proteins by simply replacing recognition elements of proximity probes.

  16. Interaction of KRAS G-quadruplex with natural polyphenols: A spectroscopic analysis with molecular modeling.

    Science.gov (United States)

    Pattanayak, Rudradip; Basak, Pijush; Sen, Srikanta; Bhattacharyya, Maitree

    2016-08-01

    Researchers are endeavoring to find out new therapeutics for curing cancer and G-quadruplex DNA has already been identified as a prospective one in this venture. Stabilizing G-quadruplex structures of telomere has emerged to be an important strategy in this context. Mutation in KRAS is mostly responsible for pancreatic, lung and colon cancer. In this present study we explored binding and conformational behaviour of G-quadruplex with different ligands by utilizing several biophysical techniques. Natural polyphenols like Curcumin and Ellagic acid were observed to bind with the G-quadruplex and enhance the melting temperature significantly indicating higher stability. UV-vis spectroscopy confirms formation of G quadruplex-ligand complex for both the compounds with specific binding affinity. Fluorimetric studies revealed that Ellagic acid had stronger binding affinity, 1.10×10(5)M(-1) compared to Curcumin, 1.6×10(4)M(-1) towards G-quadruplex. Interestingly, Curcumin provides greater stability by stacking on the top of the quadruplex structure with the help of the loops compared to Ellagic acid as is evident by docking studies. The keto form of curcumin showed stronger affinity than the enol form. We have developed a general model to estimate the influence of the ligands towards stabilizing the G-quadruplex subsequently characterizing the binding profile to enlighten prospective therapeutics.

  17. G-quadruplex DNAzymes-induced highly selective and sensitive colorimetric sensing of free heme in rat brain.

    Science.gov (United States)

    Li, Ruimin; Jiang, Qin; Cheng, Hanjun; Zhang, Guoqiang; Zhen, Mingming; Chen, Daiqin; Ge, Jiechao; Mao, Lanqun; Wang, Chunru; Shu, Chunying

    2014-04-21

    Direct selective determination of free heme in the cerebral system is of great significance due to the crucial roles of free heme in physiological and pathological processes. In this work, a G-quadruplex DNAzymes-induced highly sensitive and selective colorimetric sensing of free heme in rat brain is established. Initially, the conformation of an 18-base G-rich DNA sequence, PS2.M (5'-GTGGGTAGGGCGGGTTGG-3'), in the presence of K(+), changes from a random coil to a "parallel" G-quadruplex structure, which can bind free heme in the cerebral system with high affinity through π-π stacking. The resulted heme/G-quadruplex complex exhibits high peroxidase-like activity, which can be used to catalyze the oxidation of colorless ABTS(2-) to green ABTS˙(-) by H2O2. The concentration of heme can be evaluated by the naked eye and determined by UV-vis spectroscopy. The signal output showed a linear relationship for heme within the concentration range from 1 to 120 nM with a detection limit of 0.637 nM. The assay demonstrated here was highly selective and free from the interference of physiologically important species such as dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbate acid (AA), cysteine, uric acid (UA), glucose and lactate in the cerebral system. The basal dialysate level of free heme in the microdialysate from the striatum of adult male Sprague-Dawley rats was determined to be 32.8 ± 19.5 nM (n = 3). The analytic protocol possesses many advantages, including theoretical simplicity, low-cost technical and instrumental demands, and responsible detection of heme in rat brain microdialysate.

  18. Development of a universal colorimetric indicator for G-quadruplex structures by the fusion of thiazole orange and isaindigotone skeleton.

    Science.gov (United States)

    Yan, Jin-Wu; Ye, Wen-Jie; Chen, Shuo-Bin; Wu, Wei-Bin; Hou, Jin-Qiang; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu

    2012-08-07

    The rapid and convenient method for identification of all kinds of G-quadruplex is highly desirable. In the present study, a novel colorimetric indicator for a vast variety of G-quadruplex was designed and synthesized on the basis of thiazole orange and isaindigotone skeleton. Its distinct color change enables label-free visual detection of G-quadruplexes, which is due to the disassembly of dye H-aggregates to monomers. This specific detection of G-quadruplex arises from its end-stacking interaction with G-quartet. On the basis of this universal indicator, a facile approach for large-scale identification of G-quadruplex was developed.

  19. Binding Studies of Natural Product Berberine with DNA G-Quadruplex

    Directory of Open Access Journals (Sweden)

    Nagendra K. Sharma

    2011-01-01

    Full Text Available Problem statement: The ends of chromosome had highly repetitive short G and C-rich sequences of DNA. These sequences were known to form stable tetraplex type of secondary structures which help to maintain gene integratity after cell divison. Approach: Any reagent which controls the random cell division would be useful to design anticancer drugs. Therefore a many natural and synthesized molecules which stabilized tetraplex structures are targeted as anticancer drug entities. Results: Among them, Berberine hydrochloride natural product and its analogues are well studies as G-quadruplex stabilizing agent. In this report, DNA sequence 5’-G3-C5-G3-3’ has been designed which has probability to form i-motif and G-qua druplex types of secondary structures. Herein we studied the interaction between this DNA strands and Berberine hydrochloride by 1H-NMR techniques and UV in two different PH (4.7 and 7.4 conditions. Conclusion/Recommendations: Our preliminary results showed that Berberine bind with this DNA strand in both pH conditions which is further supported by UV melting experiments. In future this sequence can be used as probe to screen out tetraplex binding natural products which help to generate new anticancer drugs.

  20. G-quadruplex based Exo III-assisted signal amplification aptasensor for the colorimetric detection of adenosine.

    Science.gov (United States)

    Xu, Lei; Shen, Xin; Li, Bingzhi; Zhu, Chunhong; Zhou, Xuemin

    2017-08-08

    Adenosine is an endogenous nucleotide pivotally involved in nucleic acid and energy metabolism. Its excessive existence may indicate tumorigenesis, typically lung cancer. Encouraged by its significance as the clinical biomarker, sensitive assay methods towards adenosine have been popularized, with high cost and tedious procedures as the inevitable defects. Herein, we report a label-free aptamer-based exonuclease III (Exo III) amplification colorimetric aptasensor for the highly sensitive and cost-effective detection of adenosine. The strategy employed two unlabeled hairpin DNA oligonucleotides (HP1 and HP2), where HP1 contained the aptamer towards adenosine and HP2 embedded the guanine-rich sequence (GRS). In the presence of adenosine, hairpin HP1 could form specific binding with adenosine and trigger the unfolding of HP1's hairpin structure. The resulting adenosine-HP1 complex could hybridize with HP2, generating the Exo III recognition site. After Exo III-assisted degradation, the GRS was released from HP2, and the adenosine-HP1 was released back to the solution to combine another HP2, inducing the cycling amplification. After multiple circulations, the released ample GRSs were induced to form G-quadruplex, further catalyzing the oxidation of TMB, yielding a color change which was finally mirrored in the absorbance change. On the contrary, the absence of adenosine failed to unfold HP1, remaining color unchanged eventually. Thanks to the amplification strategy, the limit of detection was lowered to 17 nM with a broad linear range from 50 nM to 6 μM. The proposed method was successfully applied to the detection of adenosine in biological samples and satisfying recoveries were acquired. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding.

    Science.gov (United States)

    Wirmer-Bartoschek, Julia; Bendel, Lars Erik; Jonker, Hendrik R A; Grün, J Tassilo; Papi, Francesco; Bazzicalupi, Carla; Messori, Luigi; Gratteri, Paola; Schwalbe, Harald

    2017-06-12

    Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Quinolino[3,4-b]quinoxalines and pyridazino[4,3-c]quinoline derivatives: Synthesis, inhibition of topoisomerase IIα, G-quadruplex binding and cytotoxic properties.

    Science.gov (United States)

    Palluotto, Fausta; Sosic, Alice; Pinato, Odra; Zoidis, Grigoris; Catto, Marco; Sissi, Claudia; Gatto, Barbara; Carotti, Angelo

    2016-11-10

    The quinoline motif fused with other heterocyclic systems plays an important role in the field of anticancer drug development. An extensive series of tetracyclic quinolino[3,4-b]quinoxalines N-5 or C-6 substituted with basic side chain and a limited number of tricyclic pyridazino[4,3-c]quinolines N-6 substituted were designed, synthesized and evaluated for topoisomerase IIα (Topo IIα) inhibitory activity, ability to bind and stabilize G-quadruplex structures and cytotoxic properties against two human cancer cell lines (HeLa and MCF-7). Almost all of the tested agents showed a high activity as Topo IIα inhibitors and G-quadruplex stabilizers. Among all the derivatives studied, the quinolino[3,4-b]quinoxalines 11 and 23, N-5 and C-6 substituted respectively, stand out as the most promising compounds. Derivative 11 resulted a selective binder to selected G-quadruplex sequences, while derivative 23 displayed the most interesting Topo IIα inhibitory activity (IC50 = 5.14 μM); both showed high cytotoxic activity (IC50 HeLa = 2.04 μM and 2.32 μM, respectively).

  3. 基于T-Hg2+-T及G四聚体自身熄灭能力的"Turn on"型单标记DNA荧光探针用于碘离子的检测%Single-labeled "Turn on" Fluorescent Oligonucleotide Probe for Iodide Detection Based on T-Hg2+-T and Inherent Quenching Ability of G-quadruplex

    Institute of Scientific and Technical Information of China (English)

    朱颖; 刘沛; 羊小海; 何磊良; 李清照; 王青; 王柯敏; 黄晋; 刘剑波

    2012-01-01

    A simple and "Turn on" type iodide anion assay method was developed, based on the inherent quenching ability of G-quadruplex and fidelity of the " thymine-Hg2+ -thymine" binding motif. A T-rich sequence was labeled with 6-carboxyfluorescein(FAM) at its 5'-end, nearby the 3'-end was a G-rich sequence which could form G-quadruplex structure instead of traditional quenchers. Upon addition of Hg2+, T-rich sequence folded into a hairpin structure which led the G-quadruplex near to the FAM, and the FAM was quenched by the G-quadruplex owning to photoinduced electron transfer between the dye and the G-quadruplex. After the addition of iodide, the fluorescence of FAM recovered considering that iodide could bind with Hg2+. The novel method for the determination of iodide was developed in linear range of 50—500 nmol/L, with the detection limit(3σ) of 30 nmol/L. This proposed method was highly selective and other anions have no interfering effects on the determination, which was successfully applied for analysis of real samples with the recovery from 92%-109% , and the RSD<4% (n=4).%利用G四聚体可以熄灭荧光的特性以及T-Hg2+-T的特殊结构,发展了一种简便的"Turn on”型碘离子检测新方法.设计了一条5'端标有荧光基团的富T序列,3'端采用能形成G四聚体的富G序列代替传统的熄灭基团.加入汞离子后,富T序列形成T-Hg2+-T机构发生折叠,G四聚体靠近荧光基团,发生光诱导电子转移,使荧光被熄灭.若加入碘离子,碘离子会与汞离子形成较稳定的配合物,汞离子从DNA上被竞争下来,探针的荧光得以恢复,且荧光强度与50 ~ 500 nmol/L的碘离子呈良好线性关系,检出限为30 nmol/L.本方法选择性好,10倍于碘离子浓度的其它常见阴离子干扰较小.检测自来水样中碘离子的回收率为92%~ 109%,相对标准偏差RSD<4%(n=4).

  4. A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

    Science.gov (United States)

    Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

    2016-06-15

    The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis.

  5. Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands.

    Science.gov (United States)

    Nielsen, Mads Corvinius; Larsen, Anders Foller; Abdikadir, Faisal Hussein; Ulven, Trond

    2014-01-24

    G-quadruplex (G4) ligands are currently receiving considerable attention as potential anticancer therapeutics. A series of phenanthroline-2,9-bistriazoles carrying tethered positive end groups has been synthesized and evaluated as G4 stabilizers. The compounds were efficiently assembled by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in CH2Cl2 and water in the presence of a complexing agent. Characterization of the target compounds on telomeric and c-KIT G4 sequences led to the identification of guanidinium-substituted compounds as potent G4 DNA ligands with high selectivity over duplex DNA. The diisopropylguanidium ligands exhibited high selectivity for the proto-oncogenic sequence c-KIT over the human telomeric sequence in the surface plasmon resonance (SPR) assay, whereas the compounds appeared potent on both G4 structures in the FRET melting temperature assay. The phenanthroline-2,9-bistriazole ligands were thus identified as potent G4 ligands with high selectivity over duplex DNA, and preliminary results indicate that the scaffold may form basis for the development of subtype-specific G4 ligands.

  6. Mapping the affinity landscape of Thrombin-binding aptamers on 2'F-ANA/DNA chimeric G-Quadruplex microarrays.

    Science.gov (United States)

    Lietard, Jory; Abou Assi, Hala; Gómez-Pinto, Irene; González, Carlos; Somoza, Mark M; Damha, Masad J

    2017-01-18

    In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2'-Fluoroarabinonucleic acid (2'F-ANA) is a prime candidate for such use in microarrays. Indeed, 2'F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2'F-ANA and 2'F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2'F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2'F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2'F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2'F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays.

  7. G-quadruplex fluorescence sensing by core-extended naphthalene diimides.

    Science.gov (United States)

    Zuffo, Michela; Doria, Filippo; Botti, Silvia; Bergamaschi, Greta; Freccero, Mauro

    2017-05-01

    Fluorescent sensing of G-quadruplex nucleic acids (G4s) is an effective strategy to elucidate their role in vitro and in vivo. Small molecule ligands have often been exploited, producing an emission light up upon binding. Naphthalene diimides (NDIs), although potent G4 binders exhibiting red-NIR fluorophores, have only been marginally exploited, as they are usually quenched upon binding. Contrary, aggregating core-extended naphthalene diimides (cex-NDIs) proved to be effective probes. We prepared a library of eighteen cex-NDIs by organic synthesis, characterising their aggregation-dependent absorption and emission properties. Absorption and emission titrations, fluorescent intercalator displacement assay (FID) and circular dichroism (CD) analysis were performed to elucidate their behavior as G4 fluorescent sensors, selectivity and binding mode. cex-NDIs aggregate under aqueous solvents and as a result, their fluorescence is mostly quenched under physiological conditions. Upon G4 binding, they disaggregate into binding monomers, producing a fluorescent light-up with anti-parallel and hybrid G4s. Contrary, with parallel G4s a light-off was recorded. For the formers a groove-like interaction was inferred by ICD signals, while for the latter an end-stacking interaction mode was hypothesized by G4-FID data. cex-NDIs G4 sensing mechanism works via a induced disaggregation. The emission response depends on the G4 topology, which dictates the prevailing -groove or end-stacking- binding mode. This study highlights the potential of cex-NDIs as G4 fluorescent probes. Besides being readily synthesized and conveniently emitting above 600nm, they light-up upon binding to anti-parallel and hybrid G4, complementing a number of other probes' selectivity for the parallel topology. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Transfer in motor sequence learning: effects of practice schedule and sequence context

    OpenAIRE

    Diana Margit Müssgens; Fredrik eUllén

    2015-01-01

    Transfer (i.e., the application of a learned skill in a novel context) is an important and desirable outcome of motor skill learning. While much research has been devoted to understanding transfer of explicit skills the mechanisms of skill transfer after incidental learning remain poorly understood. The aim of this study was to 1) examine the effect of practice schedule on transfer and 2) investigate whether sequence-specific knowledge can transfer to an unfamiliar sequence context. We traine...

  9. Charge conduction properties of a parallel-stranded DNA G-quadruplex: implications for chromosomal oxidative damage.

    Science.gov (United States)

    Huang, Yu Chuan; Cheng, Alan K H; Yu, Hua-Zhong; Sen, Dipankar

    2009-07-28

    The charge-flow properties and concomitant guanine damage patterns of a number of intermolecular and wholly parallel-stranded DNA G-quadruplexes were investigated. The DNA constructs were structurally well-defined and consisted of the G-quadruplex sandwiched and stacked between two Watson-Crick base-paired duplexes. Such duplex-quadruplex-duplex constructs were designed to minimize torsional stress as well as steric crowding at the duplex-quadruplex junctions. When anthraquinone was used to induce charge flow within the constructs, it was found that the quadruplex served both as a sink and as a moderately good conductor of electron holes, relative to DNA duplexes. Most strikingly, the quadruplex suffered very little charge-flow generated oxidative damage relative to guanines in the duplex regions and, indeed, to guanines in antiparallel quadruplexes reported in prior studies. It is likely that these differences result from a combination of steric and electronic factors. A biological conclusion that may be drawn from these data is that if, as anticipated, G-quadruplex structures form in vivo at the telomeres and other loci in eukaryotic chromosomes, their ability to serve as protective sinks against chromosomal oxidative damage may depend on their specific character and topology. From a separate perspective, our results on the conduction properties of duplex-quadruplex-duplex DNA composites suggest the utility of G-quadruplexes as junction modules in the construction of DNA-based biosensors and nanocircuitry.

  10. A Toolbox for Predicting G-Quadruplex Formation and Stability

    Directory of Open Access Journals (Sweden)

    Han Min Wong

    2010-01-01

    Full Text Available G-quadruplexes are four stranded nucleic acid structures formed around a core of guanines, arranged in squares with mutual hydrogen bonding. Many of these structures are highly thermally stable, especially in the presence of monovalent cations, such as those found under physiological conditions. Understanding of their physiological roles is expanding rapidly, and they have been implicated in regulating gene transcription and translation among other functions. We have built a community-focused website to act as a repository for the information that is now being developed. At its core, this site has a detailed database (QuadDB of predicted G-quadruplexes in the human and other genomes, together with the predictive algorithm used to identify them. We also provide a QuadPredict server, which predicts thermal stability and acts as a repository for experimental data from all researchers. There are also a number of other data sources with computational predictions. We anticipate that the wide availability of this information will be of use both to researchers already active in this exciting field and to those who wish to investigate a particular gene hypothesis.

  11. A toolbox for predicting g-quadruplex formation and stability.

    Science.gov (United States)

    Wong, Han Min; Stegle, Oliver; Rodgers, Simon; Huppert, Julian Leon

    2010-01-01

    G-quadruplexes are four stranded nucleic acid structures formed around a core of guanines, arranged in squares with mutual hydrogen bonding. Many of these structures are highly thermally stable, especially in the presence of monovalent cations, such as those found under physiological conditions. Understanding of their physiological roles is expanding rapidly, and they have been implicated in regulating gene transcription and translation among other functions. We have built a community-focused website to act as a repository for the information that is now being developed. At its core, this site has a detailed database (QuadDB) of predicted G-quadruplexes in the human and other genomes, together with the predictive algorithm used to identify them. We also provide a QuadPredict server, which predicts thermal stability and acts as a repository for experimental data from all researchers. There are also a number of other data sources with computational predictions. We anticipate that the wide availability of this information will be of use both to researchers already active in this exciting field and to those who wish to investigate a particular gene hypothesis.

  12. Selective recognition and stabilization of new ligands targeting the potassium form of the human telomeric G-quadruplex DNA

    Science.gov (United States)

    Lin, Yi-Hwa; Chuang, Show-Mei; Wu, Pei-Ching; Chen, Chun-Liang; Jeyachandran, Sivakamavalli; Lo, Shou-Chen; Huang, Hsu-Shan; Hou, Ming-Hon

    2016-08-01

    The development of a ligand that is capable of distinguishing among the wide variety of G-quadruplex structures and targeting telomeres to treat cancer is particularly challenging. In this study, the ability of two anthraquinone telomerase inhibitors (NSC749235 and NSC764638) to target telomeric G-quadruplex DNA was probed. We found that these ligands specifically target the potassium form of telomeric G-quadruplex DNA over the DNA counterpart. The characteristic interaction with the telomeric G-quadruplex DNA and the anticancer activities of these ligands were also explored. The results of this present work emphasize our understanding of the binding selectivity of anthraquinone derivatives to G-quadruplex DNA and assists in future drug development for G-quadruplex-specific ligands.

  13. Studies of G-quadruplex DNA structures at the single molecule level

    DEFF Research Database (Denmark)

    Kragh, Sofie Louise

    2015-01-01

    Folding of G-quaduplex structures adopted by the human telomeric repeat is here studied by single molecule FRET microscopy. This method allows for the investigation of G-quadruplex structures and their conformational dynamic. Telomeres are located at the ends of our chromosomes and end in a single...... populations and thus providing more information than traditional ensemble experiments. Using single molecule FRET microscopy different aspects of G-quadruplex folding were investigated. We have obtained direct insight into G-quadruplex structural polymorphism both in K+ and Na+ solutions. Polymorphism have...... previously only been investigated in K+. Here, we observe significant polymorphism also in Na+. By investigating the dynamics of these conformational changes and comparing these findings with other experiments for G-quadruplexes with known topology we are able to identify different conformations and folding...

  14. Different Sequences of Feedback Types: Effectiveness, Attitudes, and Preferences

    Science.gov (United States)

    Wanchid, Raveewan

    2015-01-01

    The purposes of this research were to: 1) to compare the effects of different sequences of feedback types on the students' writing ability and their effect size; 2) to compare the effects of the levels of general English proficiency (high, moderate, and low) on the students' writing ability and their effect size; 3) to investigate the interaction…

  15. The effect of single nucleotide polymorphisms in G-rich regions of high-risk human papillomaviruses on structural diversity of DNA.

    Science.gov (United States)

    Marušič, Maja; Hošnjak, Lea; Krafčikova, Petra; Poljak, Mario; Viglasky, Viktor; Plavec, Janez

    2017-05-01

    Infection with high-risk human papillomaviruses (HPVs) can lead to development of cancer of the head and neck and anogenital regions. G-rich sequences found in genomes of high-risk HPVs can fold into non-canonical secondary structures that could serve as 3D motifs distinct from double-stranded DNA and present recognition sites for ligands and opportunity for gene expression modulation. Combination of UV, CD and NMR spectroscopy and PAGE electrophoresis were used as they offer complementary insights into structural changes of G-rich oligonucleotides. G-rich region of HPV16 is shown to preferentially form hairpin structures, while regions of HPV18, HPV52 and HPV58 fold into four-stranded DNA structures called G-quadruplexes. Single nucleotide polymorphisms found in G-rich sequences have been found to promote formation of hairpin structures of HPV16 and have affected number of species formed in G-rich region of HPV52, whereas they have exhibited minimal effect on the formation of HPV18 and HPV58 G-quadruplex structures. These structural changes were reflected in differences in apparent thermal stabilities. Potential of G-rich sequences as drug targets was evaluated based on the results of the current study. HPV16 and HPV18 are considered less appropriate targets due to several single nucleotide polymorphisms and low stability, respectively. On the other hand, HPV52 and HPV58 could be used for small-molecule mediated stabilization. G-rich sequences occurring in high-risk HPVs can fold into hairpin and G-quadruplex structures that could be potentially utilized as drug targets. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Fully integrated graphene electronic biosensor for label-free detection of lead (II) ion based on G-quadruplex structure-switching.

    Science.gov (United States)

    Li, Yijun; Wang, Cheng; Zhu, Yibo; Zhou, Xiaohong; Xiang, Yu; He, Miao; Zeng, Siyu

    2017-03-15

    This work presents a fully integrated graphene field-effect transistor (GFET) biosensor for the label-free detection of lead ions (Pb(2+)) in aqueous-media, which first implements the G-quadruplex structure-switching biosensing principle in graphene nanoelectronics. We experimentally illustrate the biomolecular interplay that G-rich DNA single-strands with one-end confined on graphene surface can specifically interact with Pb(2+) ions and switch into G-quadruplex structures. Since the structure-switching of electrically charged DNA strands can disrupt the charge distribution in the vicinity of graphene surface, the carrier equilibrium in graphene sheet might be altered, and manifested by the conductivity variation of GFET. The experimental data and theoretical analysis show that our devices are capable of the label-free and specific quantification of Pb(2+) with a detection limit down to 163.7ng/L. These results first verify the signaling principle competency of G-quadruplex structure-switching in graphene electronic biosensors. Combining with the advantages of the compact device structure and convenient electrical signal, a label-free GFET biosensor for Pb(2+) monitoring is enabled with promising application potential.

  17. Design, synthesis and evaluation of 4,7-diamino-1,10-phenanthroline G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, Mads Corvinius; Borch, Jonas; Ulven, Trond

    2009-01-01

    the central ionic column. Introduction of positively charged side chains results in compounds with appreciable G-quadruplex stabilizing properties and high aqueous solubility, with the longer side chains giving more potent compounds. Ligands carrying guanidine side chains in general show higher quadruplex...... stabilizing activity and distinctly slower kinetic properties than their amino and dimethylamino analogues, possibly due to specific hydrogen bond interactions with the G-quadruplex loops....

  18. Computational and Experimental Characterization of Ribosomal DNA and RNA G-Quadruplexes

    Science.gov (United States)

    Cho, Samuel

    DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. Recent studies strongly suggest that guanine (G)-rich genes encoding pre-ribosomal RNA (pre-rRNA) are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis. However, the structures of ribosomal G-quadruplexes at atomic resolution are unknown, and very little biophysical characterization has been performed on them to date. Here, we have modeled two putative rDNA G-quadruplex structures, NUC 19P and NUC 23P, which we observe via circular dichroism (CD) spectroscopy to adopt a predominantly parallel topology, and their counterpart rRNA. To validate and refine the putative ribosomal G-quadruplex structures, we performed all-atom molecular dynamics (MD) simulations using the CHARMM36 force field in the presence and absence of stabilizing K + or Na + ions. We optimized the CHARMM36 force field K + parameters to be more consistent with quantum mechanical calculations (and the polarizable Drude model force field) so that the K + ion is predominantly in the G-quadruplex channel. Our MD simulations show that the rDNA G-quadruplex have more well-defined, predominantly parallel-topology structures than rRNA and NUC 19P is more structured than NUC 23P, which features extended loops. Our study demonstrates that they are both potential targets for the design of novel chemotherapeutics.

  19. The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons.

    Science.gov (United States)

    Moruno-Manchon, Jose F; Koellhoffer, Edward C; Gopakumar, Jayakrishnan; Hambarde, Shashank; Kim, Nayun; McCullough, Louise D; Tsvetkov, Andrey S

    2017-09-12

    The G-quadruplex is a non-canonical DNA secondary structure formed by four DNA strands containing multiple runs of guanines. G-quadruplexes play important roles in DNA recombination, replication, telomere maintenance, and regulation of transcription. Small molecules that stabilize the G-quadruplexes alter gene expression in cancer cells. Here, we hypothesized that the G-quadruplexes regulate transcription in neurons. We discovered that pyridostatin, a small molecule that specifically stabilizes G-quadruplex DNA complexes, induced neurotoxicity and promoted the formation of DNA double-strand breaks (DSBs) in cultured neurons. We also found that pyridostatin downregulated transcription of the Brca1 gene, a gene that is critical for DSB repair. Importantly, in an in vitro gel shift assay, we discovered that an antibody specific to the G-quadruplex structure binds to a synthetic oligonucleotide, which corresponds to the first putative G-quadruplex in the Brca1 gene promoter. Our results suggest that the G-quadruplex complexes regulate transcription in neurons. Studying the G-quadruplexes could represent a new avenue for neurodegeneration and brain aging research.

  20. G-Quadruplex in the NRF2 mRNA 5' Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress.

    Science.gov (United States)

    Lee, Sang C; Zhang, Jack; Strom, Josh; Yang, Danzhou; Dinh, Thai Nho; Kappeler, Kyle; Chen, Qin M

    2017-01-01

    Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did posttranslational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress. Copyright © 2016 American Society for Microbiology.

  1. A facile label-free G-quadruplex based fluorescent aptasensor method for rapid detection of ATP

    Science.gov (United States)

    Liu, Haisheng; Ma, Changbei; Ning, Feng; Chen, Hanchun; He, Hailun; Wang, Kemin; Wang, Jun

    2017-03-01

    The present work demonstrates a simple, rapid and label-free ATP detection method using a fluorescent aptasensor that is based on G-quadruplex formation. In the absence of ATP, the Thioflavin T (ThT) dye binds to the G-rich ATP aptamer and forms an ATP aptamer/ThT G-quadruplex complex, which results in high fluorescence intensity. Upon addition of ATP, the ATP aptamer/ThT complex will be replaced by the formation of an ATP aptamer/ATP complex. During this process, separation of the ThT dye from the ATP aptamer/ThT complex decreases the fluorescence intensity of the reaction mixture dramatically. This fluorescence aptasensor is highly sensitive and rapid, with a detection limit of 18 nM and a total reaction time of only 10 min. Furthermore, this method is cost-effective and simple, removing the requirement for labeling the detection reagents with a fluorophore-quencher pair.

  2. The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

    Science.gov (United States)

    Lyonnais, Sébastien; Tarrés-Soler, Aleix; Rubio-Cosials, Anna; Cuppari, Anna; Brito, Reicy; Jaumot, Joaquim; Gargallo, Raimundo; Vilaseca, Marta; Silva, Cristina; Granzhan, Anton; Teulade-Fichou, Marie-Paule; Eritja, Ramon; Solà, Maria

    2017-01-01

    The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria. PMID:28276514

  3. Aven recognition of RNA G-quadruplexes regulates translation of the mixed lineage leukemia protooncogenes.

    Science.gov (United States)

    Thandapani, Palaniraja; Song, Jingwen; Gandin, Valentina; Cai, Yutian; Rouleau, Samuel G; Garant, Jean-Michel; Boisvert, Francois-Michel; Yu, Zhenbao; Perreault, Jean-Pierre; Topisirovic, Ivan; Richard, Stéphane

    2015-08-12

    G-quadruplexes (G4) are extremely stable secondary structures forming stacks of guanine tetrads. DNA G4 structures have been extensively studied, however, less is known about G4 motifs in mRNAs, especially in their coding sequences. Herein, we show that Aven stimulates the mRNA translation of the mixed lineage leukemia (MLL) proto-oncogene in an arginine methylation-dependent manner. The Aven RGG/RG motif bound G4 structures within the coding regions of the MLL1 and MLL4 mRNAs increasing their polysomal association and translation, resulting in the induction of transcription of leukemic genes. The DHX36 RNA helicase associated with the Aven complex and was required for optimal translation of G4 mRNAs. Depletion of Aven led to a decrease in synthesis of MLL1 and MLL4 proteins resulting in reduced proliferation of leukemic cells. These findings identify an Aven-centered complex that stimulates the translation of G4 harboring mRNAs, thereby promoting survival of leukemic cells.

  4. G-Quadruplexes Light up Localized DNA Circuits.

    Science.gov (United States)

    Mendoza, Oscar; Mergny, Jean-Louis; Aimé, Jean-Pierre; Elezgaray, Juan

    2016-01-13

    DNA circuits tethered to nanoplatforms can perform cascade reactions for signal amplification. One DNA single strand activates a strand-displacement cascade generating numerous outputs, and therefore amplifying the signal. These localized circuits present, however, an important limitation: the spontaneous activation of the cascade reaction. Current methods to stabilize these circuits employ combination of protective DNA strands, which need to be removed to activate the device. This protection-deprotection process generates an important amount of unwanted side reactions. This is indeed an important limitation for the large potential application of these amplification circuits. In the present work, G-quadruplex DNA structures were used to stabilize localized DNA circuits. This new protocol generates nanoplatforms that no longer requires protective-deprotective systems and is therefore completely neutral to the sample. In addition, cations such as Pb(2+) or Ca(2+) can be also employed to activate the device enlarging the potential applications from biosensors devices to metal detector sensors.

  5. Short sequence effect of ancient DNA on mammoth phylogenetic analyses

    Institute of Scientific and Technical Information of China (English)

    Guilian SHENG; Lianjuan WU; Xindong HOU; Junxia YUAN; Shenghong CHENG; Bojian ZHONG; Xulong LAI

    2009-01-01

    The evolution of Elephantidae has been intensively studied in the past few years, especially after 2006. The molecular approaches have made great contribution to the assumption that the extinct woolly mammoth has a close relationship with the Asian elephant instead of the African elephant. In this study, partial ancient DNA sequences of cytochrome b (cyt b) gene in mitochondrial genome were successfully retrieved from Late Pleistocene Mammuthus primigenius bones collected from Heilongjiang Province in Northeast China. Both the partial and complete homologous cyt b gene sequences and the whole mitochondrial genome sequences extracted from GenBank were aligned and used as datasets for phylogenetic analyses. All of the phylogenetic trees, based on either the partial or the complete cyt b gene, reject the relationship constructed by the whole mitochondrial genome, showing the occurrence of an effect of sequence length of cyt b gene on mammoth phylogenetic analyses.

  6. Xanthine and 8-oxoguanine in G-quadruplexes: formation of a G·G·X·O tetrad.

    Science.gov (United States)

    Cheong, Vee Vee; Heddi, Brahim; Lech, Christopher Jacques; Phan, Anh Tuân

    2015-12-02

    G-quadruplexes are four-stranded structures built from stacked G-tetrads (G·G·G·G), which are planar cyclical assemblies of four guanine bases interacting through Hoogsteen hydrogen bonds. A G-quadruplex containing a single guanine analog substitution, such as 8-oxoguanine (O) or xanthine (X), would suffer from a loss of a Hoogsteen hydrogen bond within a G-tetrad and/or potential steric hindrance. We show that a proper arrangement of O and X bases can reestablish the hydrogen-bond pattern within a G·G·X·O tetrad. Rational incorporation of G·G·X·O tetrads in a (3+1) G-quadruplex demonstrated a similar folding topology and thermal stability to that of the unmodified G-quadruplex. pH titration conducted on X·O-modified G-quadruplexes indicated a protonation-deprotonation equilibrium of X with a pKa ∼6.7. The solution structure of a G-quadruplex containing a G·G·X·O tetrad was determined, displaying the same folding topology in both the protonated and deprotonated states. A G-quadruplex containing a deprotonated X·O pair was shown to exhibit a more electronegative groove compared to that of the unmodified one. These differences are likely to manifest in the electronic properties of G-quadruplexes and may have important implications for drug targeting and DNA-protein interactions.

  7. Analysis of the Fragile X mental retardation protein isoforms 1, 2 and 3 interactions with the G-quadruplex forming semaphorin 3F mRNA.

    Science.gov (United States)

    Evans, Timothy L; Blice-Baum, Anna C; Mihailescu, Mihaela-Rita

    2012-02-01

    Fragile X syndrome, the most prevalent inheritable mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP) expression. FMRP is an RNA-binding protein with nucleo-cytoplasmic shuttle activity, proposed to act as a translation regulator of specific mRNAs in the brain. It has been shown that FMRP uses its arginine-glycine-glycine (RGG) box domain to bind a subset of mRNA targets that form a G-quadruplex structure. FMRP has also been shown to undergo the post-translational modifications of arginine methylation and phosphorylation, as well as alternative splicing, resulting in multiple isoforms. The alternative splice isoforms investigated in this study, isoform 1 (ISO1), isoform 2 (ISO2), and isoform 3 (ISO3), are created by the alternative splicing acceptor site at exon 15. FMRP ISO2 and ISO3 are truncated by 12 and 13 residues, respectively, relative to the longest FMRP isoform ISO1. These truncations, which are in the close proximity of the RGG box domain, preserve the integrity of the RGG box in all three isoforms, but eliminate the in vivo phosphorylation sites, present only on FMRP ISO1. We have expressed and purified recombinant FMRP ISO1, ISO2 and ISO3 in Escherichia coli, free of post-translational modifications, and by using fluorescence spectroscopy, we show that each FMRP isoform binds G-quadruplex RNA, albeit with different binding affinities, suggesting that naturally occurring sequence modifications in the proximity of the RGG box modulate its G-quadruplex RNA binding ability.

  8. Assembly of supramolecular DNA complexes containing both G-quadruplexes and i-motifs by enhancing the G-repeat-bearing capacity of i-motifs

    Science.gov (United States)

    Cao, Yanwei; Gao, Shang; Yan, Yuting; Bruist, Michael F.; Wang, Bing; Guo, Xinhua

    2017-01-01

    The single-step assembly of supramolecular complexes containing both i-motifs and G-quadruplexes (G4s) is demonstrated. This can be achieved because the formation of four-stranded i-motifs appears to be little affected by certain terminal residues: a five-cytosine tetrameric i-motif can bear ten-base flanking residues. However, things become complex when different lengths of guanine-repeats are added at the 3′ or 5′ ends of the cytosine-repeats. Here, a series of oligomers d(XGiXC5X) and d(XC5XGiX) (X = A, T or none; i < 5) are designed to study the impact of G-repeats on the formation of tetrameric i-motifs. Our data demonstrate that tetramolecular i-motif structure can tolerate specific flanking G-repeats. Assemblies of these oligonucleotides are polymorphic, but may be controlled by solution pH and counter ion species. Importantly, we find that the sequences d(TGiAC5) can form the tetrameric i-motif in large quantities. This leads to the design of two oligonucleotides d(TG4AC7) and d(TGBrGGBrGAC7) that self-assemble to form quadruplex supramolecules under certain conditions. d(TG4AC7) forms supramolecules under acidic conditions in the presence of K+ that are mainly V-shaped or ring-like containing parallel G4s and antiparallel i-motifs. d(TGBrGGBrGAC7) forms long linear quadruplex wires under acidic conditions in the presence of Na+ that consist of both antiparallel G4s and i-motifs. PMID:27899568

  9. A random effects epidemic-type aftershock sequence model.

    Science.gov (United States)

    Lin, Feng-Chang

    2011-04-01

    We consider an extension of the temporal epidemic-type aftershock sequence (ETAS) model with random effects as a special case of a well-known doubly stochastic self-exciting point process. The new model arises from a deterministic function that is randomly scaled by a nonnegative random variable, which is unobservable but assumed to follow either positive stable or one-parameter gamma distribution with unit mean. Both random effects models are of interest although the one-parameter gamma random effects model is more popular when modeling associated survival times. Our estimation is based on the maximum likelihood approach with marginalized intensity. The methods are shown to perform well in simulation experiments. When applied to an earthquake sequence on the east coast of Taiwan, the extended model with positive stable random effects provides a better model fit, compared to the original ETAS model and the extended model with one-parameter gamma random effects.

  10. Disordering of human telomeric G-quadruplex with novel antiproliferative anthrathiophenedione.

    Directory of Open Access Journals (Sweden)

    Dmitry Kaluzhny

    Full Text Available Linear heteroareneanthracenediones have been shown to interfere with DNA functions, thereby causing death of human tumor cells and their drug resistant counterparts. Here we report the interaction of our novel antiproliferative agent 4,11-bis[(2-{[acetimido]amino}ethylamino]anthra[2,3-b]thiophene-5,10-dione with telomeric DNA structures studied by isothermal titration calorimetry, circular dichroism and UV absorption spectroscopy. New compound demonstrated a high affinity (K(ass∼10⁶ M⁻¹ for human telomeric antiparallel quadruplex d(TTAGGG₄ and duplex d(TTAGGG₄∶d(CCCTAA₄. Importantly, a ∼100-fold higher affinity was determined for the ligand binding to an unordered oligonucleotide d(TTAGGG TTAGAG TTAGGG TTAGGG unable to form quadruplex structures. Moreover, in the presence of Na+ the compound caused dramatic conformational perturbation of the telomeric G-quadruplex, namely, almost complete disordering of G-quartets. Disorganization of a portion of G-quartets in the presence of K+ was also detected. Molecular dynamics simulations were performed to illustrate how the binding of one molecule of the ligand might disrupt the G-quartet adjacent to the diagonal loop of telomeric G-quadruplex. Our results provide evidence for a non-trivial mode of alteration of G-quadruplex structure by tentative antiproliferative drugs.

  11. G-quadruplex and i-motif are mutually exclusive in ILPR double-stranded DNA.

    Science.gov (United States)

    Dhakal, Soma; Yu, Zhongbo; Konik, Ryan; Cui, Yunxi; Koirala, Deepak; Mao, Hanbin

    2012-06-06

    G-quadruplex has demonstrated its biological functions in vivo. Although G-quadruplex in single-stranded DNA (ssDNA) has been well characterized, investigation of this species in double-stranded DNA (dsDNA) lags behind. Here we use chemical footprinting and laser-tweezers-based single-molecule approaches to demonstrate that a dsDNA fragment found in the insulin-linked polymorphic region (ILPR), 5'-(ACA GGGG TGT GGGG)2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K+, and an i-motif at pH 5.5 with 100 mM Li+. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K+), a unique determination of change in the free energy of unfolding (ΔGunfold) by laser-tweezers experiments provides compelling evidence that only one species is present in each dsDNA. Under this condition, molecules containing G-quadruplex are more stable than those with i-motif. These two species have mechanical stabilities (rupture force≥17 pN) comparable to the stall force of RNA polymerases, which, from a mechanical perspective alone, could justify a regulatory mechanism for tetraplex structures in the expression of human insulin. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. Effect of dephasing on DNA sequencing via transverse electronic transport

    Energy Technology Data Exchange (ETDEWEB)

    Zwolak, Michael [Los Alamos National Laboratory; Krems, Matt [NON LANL; Pershin, Yuriy V [NON LANL; Di Ventra, Massimiliano [NON LANL

    2009-01-01

    We study theoretically the effects of dephasing on DNA sequencing in a nanopore via transverse electronic transport. To do this, we couple classical molecular dynamics simulations with transport calculations using scattering theory. Previous studies, which did not include dephasing, have shown that by measuring the transverse current of a particular base multiple times, one can get distributions of currents for each base that are distinguishable. We introduce a dephasing parameter into transport calculations to simulate the effects of the ions and other fluctuations. These effects lower the overall magnitude of the current, but have little effect on the current distributions themselves. The results of this work further implicate that distinguishing DNA bases via transverse electronic transport has potential as a sequencing tool.

  13. A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple.

    Science.gov (United States)

    Kuryavyi, V; Majumdar, A; Shallop, A; Chernichenko, N; Skripkin, E; Jones, R; Patel, D J

    2001-06-29

    The architecture of G-G-G-G tetrad-aligned DNA quadruplexes in monovalent cation solution is dependent on the directionality of the four strands, which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G-G-G-G tetrads. The smallest unimolecular G-quadruplex belongs to the d(G2NnG2NnG2NnG2) family, which has the potential to form two stacked G-tetrads linked by Nn loop connectivities. Previous studies have focused on the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), where Nn was T2 for the first and third connecting loops and TGT for the middle connecting loop. This DNA aptamer in K(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(anti)-G(syn)-G(anti) tetrads, adjacent strands which are antiparallel to each other and edge-wise connecting T2, TGT and T2 loops. We now report on the NMR-based solution structure of the d(G2T4G2CAG2GT4G2T) sequence, which differs from the thrombin-binding DNA aptamer sequence in having longer first (T4) and third (GT4) loops and a shorter (CA) middle loop. This d(G2T4G2CAG2GT4G2T) sequence in Na(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads, adjacent strands which have one parallel and one antiparallel neighbors and distinct non-edge-wise loop connectivities. Specifically, the longer first (T4) and third (GT4) loops are of the diagonal type while the shorter middle loop is of the double chain reversal type. In addition, the pair of stacked G-G-G-G tetrads are flanked on one side by a G-(T-T) triad and on the other side by a T-T-T triple. The distinct differences in strand directionalities, loop connectivities and syn/anti distribution within G-G-G-G tetrads between the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2) quadruplex reported previously, and the d(G2T4G2CAG2GT4G2T) quadruplex reported here, reinforces the polymorphic nature of higher

  14. A tutorial review for employing enzymes for the construction of G-quadruplex-based sensing platforms.

    Science.gov (United States)

    Ma, Dik-Lung; Wang, Wanhe; Mao, Zhifeng; Yang, Chao; Chen, Xiu-Ping; Lu, Jin-Jian; Han, Quan-Bin; Leung, Chung-Hang

    2016-03-24

    With rapid advances in the field of DNA chemistry, nucleic acids and DNA-modifying enzymes have recently emerged as versatile components for the construction of oligonucleotide-based sensors. Meanwhile, the G-quadruplex motif has been widely employed for the development of DNA-based assays due to its diverse structural variety. In this tutorial, we introduce the principles of G-quadruplex-based sensing and the use of DNA-modifying enzymes for sensor platform development. We also highlight recent studies of the application of DNA-modifying enzymes for the development of G-quadruplex-based luminescent detection platforms with a view towards how those enzymes play an important role in sensitivity enhancement.

  15. Improved in vitro efficacy of gold nanoconstructs by increased loading of G-quadruplex aptamer.

    Science.gov (United States)

    Dam, Duncan Hieu M; Lee, Raymond C; Odom, Teri W

    2014-05-14

    This paper describes how in vitro efficacy of aptamer-loaded gold nanostars (Apt-AuNS) can be enhanced by the increased loading of a G-quadruplex homodimer AS1411 (Apt) on the AuNS surface. In a low pH buffer environment, the loading density of Apt on AuNS was increased up to 2.5 times that obtained using the conventional salt-aging process. These highly loaded AuNS nanoconstructs (*Apt-AuNS) were taken up in pancreatic cancer and fibrosarcoma cells ca. 2 times more and at faster rates compared to Apt-AuNS. When a similar number of AuNS carriers was internalized by the cancer cells, the amount of AS1411 delivered via *Apt-AuNS was effectively double that of Apt-AuNS, and *Apt-AuNS resulted in an average of 42% increase in cell death. These results suggest that increasing the loading density on AuNS could provide a simple means to improve uptake as well as in vitro efficacy of the nanoconstructs in cancer cells.

  16. Study of STAT3 G-quadruplex folding patterns by CD spectroscopy and molecular modeling

    Institute of Scientific and Technical Information of China (English)

    Sen Lin; Ming Xu; Gu Yuan

    2012-01-01

    The G-quadruplexes formed from G-rich strands in the telomere and oncogene-promoter regions are regarded as new promising targets in the cancer therapy.A G-quadruplex in the downstream flanking region of the signal transducer and activator of transcription 3 (STAT3) gene was explored.Its folding patterns were proposed to be 3∶2∶2 and 3∶3∶1 loop isomers by the mutation analysis by CD spectroscopy.The structures were constructed and refined by molecular modeling method.

  17. Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes

    Science.gov (United States)

    Tanpure, Arun A.; Srivatsan, Seergazhi G.

    2015-01-01

    Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2′-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential. PMID:26202965

  18. Effects of sequence on DNA wrapping around histones

    Science.gov (United States)

    Ortiz, Vanessa

    2011-03-01

    A central question in biophysics is whether the sequence of a DNA strand affects its mechanical properties. In epigenetics, these are thought to influence nucleosome positioning and gene expression. Theoretical and experimental attempts to answer this question have been hindered by an inability to directly resolve DNA structure and dynamics at the base-pair level. In our previous studies we used a detailed model of DNA to measure the effects of sequence on the stability of naked DNA under bending. Sequence was shown to influence DNA's ability to form kinks, which arise when certain motifs slide past others to form non-native contacts. Here, we have now included histone-DNA interactions to see if the results obtained for naked DNA are transferable to the problem of nucleosome positioning. Different DNA sequences interacting with the histone protein complex are studied, and their equilibrium and mechanical properties are compared among themselves and with the naked case. NLM training grant to the Computation and Informatics in Biology and Medicine Training Program (NLM T15LM007359).

  19. Fluorescent Dansyl-Guanosine Conjugates that Bind c-MYC Promoter G-Quadruplex and Downregulate c-MYC Expression.

    Science.gov (United States)

    Pavan Kumar, Y; Saha, Puja; Saha, Dhurjhoti; Bessi, Irene; Schwalbe, Harald; Chowdhury, Shantanu; Dash, Jyotirmayee

    2016-03-02

    The four-stranded G-quadruplex present in the c-MYC P1 promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Small-molecule compounds capable of inhibiting the c-MYC promoter activity by stabilising the c-MYC G-quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl-guanosine conjugates through one-pot modular click reactions. The dansyl-guanosine conjugates can selectively detect c-MYC G-quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c-MYC G-quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c-MYC G-quadruplex. A dual luciferase assay and qRT-PCR revealed that a dansyl-bisguanosine ligand represses the c-MYC expression, possibly by stabilising the c-MYC G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Populated intermediates in the thermal unfolding of the human telomeric quadruplex.

    Science.gov (United States)

    Gray, Robert D; Buscaglia, Robert; Chaires, Jonathan B

    2012-10-10

    Thermal denaturation profiles of several model oligonucleotides of the human telomere DNA sequence including d[A(GGGTTA)(3)GGG] (Tel22) were determined using circular dichroism (CD), fluorescence of adenine → 2-aminopurine analogs, and fluorescence resonance energy transfer (FRET) to monitor the unfolding process at specific locations within the quadruplex. The resulting optical spectra vs temperature data matrices were analyzed by singular value decomposition (SVD) to ascertain the minimum number of species required to reproduce the unfolding spectral profiles. Global nonlinear least-squares fitting of the SVD amplitude vectors was used to estimate thermodynamic parameters and optical spectra of all species for a series of unfolding mechanisms that included one-, two-, and three-step sequential pathways F ⇌ I(n) ⇌ U, n = 0, 1, or 2) as well as two mechanisms with spectroscopically distinct starting structures (F(1) and F(2)). The CD and FRET data for Tel22 unfolding between 4 and 94 °C in 25 mM KCl were best described by a sequential unfolding model with two intermediates, while the 2-aminopurine analogs required one intermediate. The higher melting intermediate I(2) had a transition midpoint temperature (T(m)) of 61 °C and a CD spectrum with a maximum and minimum at ~265 and ~245 nm, respectively. The fluorescence emission spectra of the 2-aminopurine and FRET derivatives suggest greater solvent exposure of the 5'-AGGGTTA- segment in the intermediate compared to the folded state. The spectroscopic properties of the 61 °C intermediate suggest that it may be a triple helical structure.

  1. Effects of the Ion PGM™ Hi-Q™ sequencing chemistry on sequence data quality.

    Science.gov (United States)

    Churchill, Jennifer D; King, Jonathan L; Chakraborty, Ranajit; Budowle, Bruce

    2016-09-01

    Massively parallel sequencing (MPS) offers substantial improvements over current forensic DNA typing methodologies such as increased resolution, scalability, and throughput. The Ion PGM™ is a promising MPS platform for analysis of forensic biological evidence. The system employs a sequencing-by-synthesis chemistry on a semiconductor chip that measures a pH change due to the release of hydrogen ions as nucleotides are incorporated into the growing DNA strands. However, implementation of MPS into forensic laboratories requires a robust chemistry. Ion Torrent's Hi-Q™ Sequencing Chemistry was evaluated to determine if it could improve on the quality of the generated sequence data in association with selected genetic marker targets. The whole mitochondrial genome and the HID-Ion STR 10-plex panel were sequenced on the Ion PGM™ system with the Ion PGM™ Sequencing 400 Kit and the Ion PGM™ Hi-Q™ Sequencing Kit. Concordance, coverage, strand balance, noise, and deletion ratios were assessed in evaluating the performance of the Ion PGM™ Hi-Q™ Sequencing Kit. The results indicate that reliable, accurate data are generated and that sequencing through homopolymeric regions can be improved with the use of Ion Torrent's Hi-Q™ Sequencing Chemistry. Overall, the quality of the generated sequencing data supports the potential for use of the Ion PGM™ in forensic genetic laboratories.

  2. Sequence-Dependent Effects on the Properties of Semiflexible Biopolymers

    CERN Document Server

    Zicong, Bela

    2008-01-01

    Using path integral technique, we show exactly that for a semiflexible biopolymer in constant extension ensemble, no matter how long the polymer and how large the external force, the effects of short range correlations in the sequence-dependent spontaneous curvatures and torsions can be incorporated into a model with well-defined mean spontaneous curvature and torsion as well as a renormalized persistence length. Moreover, for a long biopolymer with large mean persistence length, the sequence-dependent persistence lengths can be replaced by their mean. However, for a short biopolymer or for a biopolymer with small persistence lengths, inhomogeneity in persistence lengths tends to make physical observables very sensitive to details and therefore less predictable.

  3. Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex dependent fashion.

    Science.gov (United States)

    Saha, Dhurjhoti; Singh, Ankita; Hussain, Tabish; Srivastava, Vivek; Sengupta, Suman; Kar, Anirban; Dhapola, Parashar; Dhople, Vishnu; Ummani, Ramesh; Chowdhury, Shantanu

    2017-07-17

    Transcriptional activation of the human telomerase reverse transcriptase (hTERT) gene, which remains repressed in adult somatic cells, is critical during tumorigenesis. Several transcription factors and the epigenetic state of the hTERT promoter are known to be important for tight control of hTERT in normal tissues, but the molecular mechanisms leading to hTERT reactivation in cancer are not well understood. Surprisingly, here we found occupancy of the metastasis suppressor nonmetastatic 2 (NME2) within hTERT core promoter in HT1080 fibrosarcoma cells and HCT116 colon cancer cells, and NME2 mediated transcriptional repression of hTERT in these cells. We also report that loss of NME2 results in upregulated hTERT expression. Mechanistically, additional results indicated that the RE1 silencing transcription factor (REST), lysine-specific histone demethylase 1 (LSD1) co-repressor complex associates with the hTERT promoter in a NME2-dependent way, and that this assembly is required for maintaining repressive chromatin at the hTERT promoter. Interestingly, a G-quadruplex motif at the hTERT promoter was essential for occupancy of NME2 and the REST repressor complex on the hTERT promoter. In light of this mechanistic insight, we studied the effects of G quadruplex binding ligands on hTERT expression and observed that several of these ligands repressed hTERT expression. Together, our results support a mechanism of hTERT epigenetic control involving a G quadruplex promoter motif, which potentially can be targeted by tailored small molecules. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  4. Effects of priming goal pursuit on implicit sequence learning.

    Science.gov (United States)

    Gamble, Katherine R; Lee, Joanna M; Howard, James H; Howard, Darlene V

    2014-11-01

    Implicit learning, the type of learning that occurs without intent to learn or awareness of what has been learned, has been thought to be insensitive to the effects of priming, but recent studies suggest this is not the case. One study found that learning in the serial reaction time (SRT) task was improved by nonconscious goal pursuit, primed via a word search task (Eitam et al. in Psychol Sci 19:261-267, 2008). In two studies, we used the goal priming word search task from Eitam et al., but with a different version of the SRT, the alternating serial reaction time task (ASRT). Unlike the SRT, which often results in explicit knowledge and assesses sequence learning at one point in time, the ASRT has been shown to be implicit through sensitive measures of judgment, and it enables sequence learning to be measured continuously. In both studies, we found that implicit learning was superior in the groups that were primed for goal achievement compared to control groups, but the effect was transient. We discuss possible reasons for the observed time course of the positive effects of goal priming, as well as some future areas of investigation to better understand the mechanisms that underlie this effect, which could lead to methods to prolong the positive effects.

  5. MutS recognition: Multiple mismatches and sequence context effects

    Indian Academy of Sciences (India)

    Amita Joshi; Basuthkar J Rao

    2001-12-01

    Escherichia coli MutS is a versatile repair protein that specifically recognizes not only various types of mismatches but also single stranded loops of up to 4 nucleotides in length. Specific binding, followed by the next step of tracking the DNA helix that locates hemi-methylated sites, is regulated by the conformational state of the protein as a function of ATP binding/hydrolysis. Here, we study how various molecular determinants of a heteroduplex regulate mismatch recognition by MutS, the critical first step of mismatch repair. Using classical DNase I footprinting assays, we demonstrate that the hierarchy of MutS binding to various types of mismatches is identical whether the mismatches are present singly or in multiples. Moreover, this unique hierarchy is indifferent both to the differential level of DNA helical flexibility and to the unpaired status of the mismatched bases in a heteroduplex. Surprisingly, multiple mismatches exhibit reduced affinity of binding to MutS, compared to that of a similar single mismatch. Such a reduction in the affinity might be due to sequence context effects, which we established more directly by studying two identical single mismatches in an altered sequence background. A mismatch, upon simply being flipped at the same location, elicits changes in MutS specific contacts, thereby underscoring the importance of sequence context in modulating MutS binding to mismatches.

  6. Rapid and simultaneous detection of common aneuploidies by quadruplex real-time polymerase chain reaction combined with melting curve analysis

    Science.gov (United States)

    Lou, Jiwu; Sun, Manna; Zhao, Ying; Ji, Zhisong; Liu, Fenghua; Li, Dongzhi; Xu, Wanfang; Lin, Yangyang; Liu, Yanhui

    2017-01-01

    Background During the prenatal period, the number variation of chromosomes 13, 18, 21, X and Y accounts for more than 80% of the clinically significant chromosomal abnormalities diagnosed. Rapid tests for prenatal diagnosis of these abnormalities can improve pregnancy management and alleviate parental anxiety. Here, we present a molecular alternative method for detecting common aneuploidies. Methods This method is based on co-amplification of segmental duplications located on two different chromosomes using a single pair of primers. Segmental duplications have a high degree of sequence identity, but have single-nucleotide differences in some regions. These sequence differences can be quantified using melting curve analysis of dual-labeled probes to estimate the relative dosages of different chromosomes. We designed two quadruplex real-time PCR assays to detect aneuploidies of chromosomes 13, 18, 21, X and Y. Results We examined 75 aneuploid DNA samples and 56 unaffected DNA control samples using these two assays and correctly identified all samples. Four cases of unbalanced translocation were also accurately detected. The observed averaged ratio for each chromosomal disorder was similar to the theoretically expected value. Conclusions Our real-time assay is a robust, rapid, and easy to conduct technique for prenatal diagnosis of common aneuploidies, representing a competitive alternative for use in diagnostic laboratories. PMID:28241016

  7. Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures

    DEFF Research Database (Denmark)

    Chatterjee, Sujoy; Zagelbaum, Jennifer; Savitsky, Pavel;

    2014-01-01

    Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA...

  8. Pentose phosphate pathway function affects tolerance to the G-quadruplex binder TMPyP4.

    Science.gov (United States)

    Andrew, Elizabeth J; Merchan, Stephanie; Lawless, Conor; Banks, A Peter; Wilkinson, Darren J; Lydall, David

    2013-01-01

    G-quadruplexes form in guanine-rich regions of DNA and the presence of these structures at telomeres prevents the activity of telomerase in vitro. Ligands such as the cationic porphyrin TMPyP4 stabilise G-quadruplexes and are therefore under investigation for their potential use as anti-cancer drugs. In order to investigate the mechanism of action of TMPyP4 in vivo, we carried out a genome-wide screen in the budding yeast Saccharomyces cerevisiae. We found that deletion of key pentose phosphate pathway (PPP) genes increased the sensitivity of yeast to the presence of TMPyP4. The PPP plays an important role in the oxidative stress response and sensitivity to TMPyP4 also increased when genes involved in the oxidative stress response, CCS1 and YAP1, were deleted. For comparison we also report genome wide-screens using hydrogen peroxide, which causes oxidative stress, RHPS4, another G-quadruplex binder and hydroxyurea, an S phase poison. We found that a number of TMPyP4-sensitive strains are also sensitive to hydrogen peroxide in a genome-wide screen. Overall our results suggest that treatment with TMPyP4 results in light-dependent oxidative stress response in budding yeast, and that this, rather than G-quadruplex binding, is the major route to cytotoxicity. Our results have implications for the usefulness and mechanism of action of TMPyP4.

  9. Spectroscopic studies of the interaction between methylene blue and G-quadruplex

    Institute of Scientific and Technical Information of China (English)

    SUN Hongxia; XIANG Junfeng; ZHANG Yazhou; XU Guangzhi; XU Lianghua; TANG Yalin

    2006-01-01

    G-rich single-stranded DNA (5'-TTAG-GG-3') adopted a G-quadruplex structure in buffer containing potassium ions. The spectroscopic feature and the interaction between methylene blue and G-quadruplex have been investigated by circular dichroism, and nuclear magnetic resonance spectroscopy. The UV-Vis absorption and fluorescence spectral results show that the fluorescence behavior of MB by single-stranded DNA fits Stern-Volmer static quenching equation very well and they formed 1:1 complexes; dimeric G-quadruplexes were bound to MB with 1:1 or 2:1, and their equilibrium constants were 1.047×105 and 8.79×104 L/mol, respectively.Based on the above results and 1H-NMR spectral data, one may conclude that MB stacked either the terminal tetrads to form 1:1 complexes or between two terminal tetrads of G-quadruplexes to form 1:2 sandwich complexes with G-qudruplexes.

  10. Tetrasubstituted phenanthrolines as highly potent, water-soluble, and selective g-quadruplex ligands

    DEFF Research Database (Denmark)

    Larsen, Anders Foller; Nielsen, Mads Corvinius; Ulven, Trond

    2012-01-01

    Small molecules capable of stabilizing the G-quadruplex (G4) structure are of interest for the development of improved anticancer drugs. Novel 4,7-diamino-substituted 1,10-phenanthroline-2,9-dicarboxamides that represent hybrid structures of known phenanthroline-based ligands have been designed. ...

  11. Design and study of telomerase inhibitors based on G-quadruplex ligands

    Directory of Open Access Journals (Sweden)

    Negrutska V. V.

    2013-05-01

    Full Text Available In this review we have summarized the results of our recent research on telomerase inhibitors and G-quadruplex DNA ligands. A series of potential enzyme inhibitors were synthesized and studied. These compounds were based on tricyclic heteroaromatic systems (thiazolobenzimidazoles phenazines, acridones, amino-substituted cyanines and natural and synthetic porphyrins and their metalocomplexes. A number of compounds, including cyanines and especially porphyrin derivatives and conjugates, were found to efficiently inhibit telomerase at low micromolar concentrations in the in vitro TRAP assay. Porphyrins demonstrated antiproliferative activity in tumor cell cultures at micro- and nanomolar concentrations. Spectral-fluorescent and electrophoretic experiments were performed to investigate the interaction of ligands with duplex and quadruplex DNA, and in many cases binding mode was established. Convenient G-octet model of G-quadruplex was developed to study the ligand-target binding using quantum-chemical methods. QM/MM hybrid approach ONIUM2 was employed to model the interaction of small molecules with Tel22 quadruplex DNA.

  12. Trigeminal star-like platinum complexes induce cancer cell senescence through quadruplex-mediated telomere dysfunction.

    Science.gov (United States)

    Zheng, Xiao-Hui; Mu, Ge; Zhong, Yi-Fang; Zhang, Tian-Peng; Cao, Qian; Ji, Liang-Nian; Zhao, Yong; Mao, Zong-Wan

    2016-12-01

    Two trigeminal star-like platinum complexes were synthesized to induce the formation of human telomere G-quadruplex (hTel G4) with extremely high selectivity and affinity. The induced hTel G4 activates strong telomeric DNA damage response (TDDR), resulting in telomere dysfunction and cell senescence.

  13. Sole and stable RNA duplexes of G-rich sequences located in the 5'-untranslated region of protooncogenes.

    Science.gov (United States)

    Saxena, Sarika; Miyoshi, Daisuke; Sugimoto, Naoki

    2010-08-24

    Guanine- (G-) rich nucleic acid sequences can form four-stranded structures called G-quadruplexes. It is widely held that the formation of a G-quadruplex in RNA is more feasible than in DNA because of the lack of a complementary strand in mRNA. Here, we analyzed sequences of 5'-untranslated regions of protooncogenes and surprisingly found that these regions showed an enrichment of not only guanine (G) but also cytosine (C) nucleotides. Since neighboring cytosine- (C-) rich regions can affect the formation and stability of a G-quadruplex structure, we further investigated the properties of DNA and RNA structures of G-rich and GC-rich regions. We selected typical GC-rich RNA sequences from protooncogenes and corresponding DNA sequences and investigated their structures. It was found that the GC-rich RNA sequences formed stable A-form duplexes as their major structure independent of the surrounding conditions, including the presence of different cations (Na(+), K(+), or Li(+)) or molecular crowding with 40 wt % poly(ethylene glycol) with an average molecular mass of 200 Da although there are a few exceptions in which only a combination of K(+) and molecular crowding induced a G-quadruplex structure of an extremely G-rich RNA sequence. In contrast, structural polymorphisms involving duplexes, G-quadruplexes, and i-motifs were observed for GC-rich DNA sequences depending on the surrounding factors. These results demonstrate the considerable structural and functional differences in GC-rich sequences of the genome (DNA) and transcriptosome (mRNA) with respect to the nucleic acid backbone. Moreover, it was suggested that structural study for a G-rich RNA sequence should be carried out under cell-mimicking condition where K(+) and crowding cosolutes exist.

  14. Hierarchical Self-Assembly of a Biomimetic Light-Harvesting Antenna Based on DNA G-Quadruplexes

    NARCIS (Netherlands)

    Oltra, Nuria Sancho; Browne, Wesley R.; Roelfes, Gerard

    2013-01-01

    A new modular approach to an artificial light-harvesting antenna system is presented. The approach involves the hierarchical self-assembly of porphyrin acceptor molecules to G-quadruplexes tethered to coumarin donor moieties.

  15. G-quadruplex recognition activities of E. Coli MutS

    Directory of Open Access Journals (Sweden)

    Ehrat Edward A

    2012-07-01

    Full Text Available Abstract Background Guanine quadruplex (G4 DNA is a four-stranded structure that contributes to genome instability and site-specific recombination. G4 DNA folds from sequences containing tandemly repetitive guanines, sequence motifs that are found throughout prokaryote and eukaryote genomes. While some cellular activities have been identified with binding or processing G4 DNA, the factors and pathways governing G4 DNA metabolism are largely undefined. Highly conserved mismatch repair factors have emerged as potential G4-responding complexes because, in addition to initiating heteroduplex correction, the human homologs bind non-B form DNA with high affinity. Moreover, the MutS homologs across species have the capacity to recognize a diverse range of DNA pairing variations and damage, suggesting a conserved ability to bind non-B form DNA. Results Here, we asked if E. coli MutS and a heteroduplex recognition mutant, MutS F36A, were capable of recognizing and responding to G4 DNA structures. We find by mobility shift assay that E. coli MutS binds to G4 DNA with high affinity better than binding to G-T heteroduplexes. In the same assay, MutS F36A failed to recognize G-T mismatched oligonucleotides, as expected, but retained an ability to bind to G4 DNA. Association with G4 DNA by MutS is not likely to activate the mismatch repair pathway because nucleotide binding did not promote release of MutS or MutS F36A from G4 DNA as it does for heteroduplexes. G4 recognition activities occur under physiological conditions, and we find that M13 phage harboring G4-capable DNA poorly infected a MutS deficient strain of E. coli compared to M13mp18, suggesting functional roles for mismatch repair factors in the cellular response to unstable genomic elements. Conclusions Taken together, our findings demonstrate that E. coli MutS has a binding activity specific for non-B form G4 DNA, but such binding appears independent of canonical heteroduplex repair activation.

  16. How effective is graphene nanopore geometry on DNA sequencing?

    CERN Document Server

    Satarifard, Vahid; Ejtehadi, Mohammad Reza

    2015-01-01

    In this paper we investigate the effects of graphene nanopore geometry on homopolymer ssDNA pulling process through nanopore using steered molecular dynamic (SMD) simulations. Different graphene nanopores are examined including axially symmetric and asymmetric monolayer graphene nanopores as well as five layer graphene polyhedral crystals (GPC). The pulling force profile, moving fashion of ssDNA, work done in irreversible DNA pulling and orientations of DNA bases near the nanopore are assessed. Simulation results demonstrate the strong effect of the pore shape as well as geometrical symmetry on free energy barrier, orientations and dynamic of DNA translocation through graphene nanopore. Our study proposes that the symmetric circular geometry of monolayer graphene nanopore with high pulling velocity can be used for DNA sequencing.

  17. Fragile X mental retardation protein recognition of G quadruplex structure per se is sufficient for high affinity binding to RNA.

    Science.gov (United States)

    Bole, Medhavi; Menon, Lakshmi; Mihailescu, Mihaela-Rita

    2008-12-01

    Fragile X syndrome, the most common form of inherited mental retardation is caused by the expansion of a CGG trinucleotide repeat in the fragile X mental retardation 1 (fmr1) gene. The abnormal expansion of the CGG repeat causes hypermethylation and subsequent silencing of the fmr1 gene, resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine rich region (RGG box) to bind to messenger RNAs that form G quadruplex structures. Several studies reported that the G quadruplex RNA recognition alone is not sufficient for FMRP RGG box binding and that an additional stem and/or a G quadruplex-stem junction region may also be important in recognition. In this study we have used biophysical methods such as fluorescence, UV, CD and NMR spectroscopy to demonstrate that the recognition of the RNA G quadruplex structure per se, in the absence of a stem region, is sufficient for the FMRP high affinity and specific binding. These findings indicate that the presence of a stem structure in some of the FMRP G quadruplex forming mRNAs is not a requirement for protein recognition as previously believed, but rather for the proper formation of the correct RNA G quadruplex structure recognized by FMRP.

  18. Spectroscopic investigation on the interaction of copper porphyrazines and phthalocyanine with human telomeric G-quadruplex DNA.

    Science.gov (United States)

    Hassani, Leila; Hakimian, Fatemeh; Safaei, Elham

    2014-01-01

    The G-quadruplex DNA is a novel target for anticancer drug discovery and many scientific groups are investigating interaction of small molecules with G-quadruplex DNA to discover therapeutic agents for cancer. Here, interaction of a phthalocyanine (Cu(PcTs)) and two tetrapyridinoporphyrazines ([Cu(2,3-tmtppa)](4+) and [Cu(3,4-tmtppa)](4+)) with Na(+) and K(+) forms of human telomeric G-quadruplex DNA has been investigated by spectroscopic techniques. The results indicated that interaction of the cationic porphyrazines is remarkably stronger than the anionic phthalocyanine and they presumably bind to the G-quadruplex DNA through end-stacking. Fluorescent intercalator displacement assay implied the displacement ability of the complexes with thiazole orange. In addition, circular dichroism spectra of both quadruplex forms converge to the Na(+) isoform after binding to the porphyrazines. In conclusion, the porphyrazines as the complexes that bind to the G-quadruplex DNA, could be suitable candidates for further investigations about inhibition of telomerase enzyme. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. In vitro HIV-1 selective integration into the target sequence and decoy-effect of the modified sequence.

    Directory of Open Access Journals (Sweden)

    Tatsuaki Tsuruyama

    Full Text Available Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5'-(GTCCCTTCCCAGT(n(ACTGGGAAGGGAC(n-3', and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA.

  20. Effects of Sequences of Socially Regulated Learning on Group Performance

    NARCIS (Netherlands)

    Molenaar, I.; Chiu, M.M.

    2015-01-01

    Past research shows that regulative activities (metacognitive or relational) can aid learning and that sequences of cognitive, metacognitive and relational activities affect subsequent cognition. Extending this research, this study examines whether sequences of socially regulated learning differ

  1. Effects of Sequences of Socially Regulated Learning on Group Performance

    NARCIS (Netherlands)

    Molenaar, I.; Chiu, M.M.

    2015-01-01

    Past research shows that regulative activities (metacognitive or relational) can aid learning and that sequences of cognitive, metacognitive and relational activities affect subsequent cognition. Extending this research, this study examines whether sequences of socially regulated learning differ acr

  2. Antiproliferative Activity of G-quadruplex Nucleic Acids%具抗肿瘤活性的G-四链体核酸研究进展

    Institute of Scientific and Technical Information of China (English)

    常天俊; 龚红梅; 李卫国

    2012-01-01

    G-四链体(G-quadruplex,G4)是由富含串联重复的鸟嘌呤碱基(G)的DNA或RNA链折叠形成的一种特殊的核酸二级结构.可形成G4结构的核酸序列在基因组和人端粒中广泛存在,对生理和病理过程起重要的调节作用.近年来研究发现,一些化学合成的G4核酸具有选择性的抗肿瘤增殖活性;其中AS1411是一个26个碱基的G4序列核酸,目前已作为抗癌药物进入二期临床研究.对G4核酸的结构和功能,抗癌活性及分子机理研究进行综合评述,并简要介绍G4核酸在相关领域的应用研究.%G-quadruplexes(G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences.G4 sequences are highly prevalent in human genome and telomere.Recently,some synthetic G4s have been reported to have cancer-selective antiproliferative activity.AS1411,a 26-mer G4 DNA,is currently in phase II clinical trials as an anticancer agent.The structures,functions,antiproliferative activities and mechanisms,and the applications of G4s are reviewed.

  3. Templated Formation of Discrete RNA and DNA:RNA Hybrid G-Quadruplexes and Their Interactions with Targeting Ligands.

    Science.gov (United States)

    Bonnat, Laureen; Dejeu, Jérôme; Bonnet, Hugues; Génnaro, Béatrice; Jarjayes, Olivier; Thomas, Fabrice; Lavergne, Thomas; Defrancq, Eric

    2016-02-24

    G-rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper-catalyzed azide-alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G-quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context.

  4. Co-barcoded sequence reads from long DNA fragments: A cost-effective solution for Perfect Genome sequencing

    Directory of Open Access Journals (Sweden)

    Brock A Peters

    2015-01-01

    Full Text Available Next generation sequencing (NGS technologies, primarily based on massively parallel sequencing (MPS, have touched and radically changed almost all aspects of research worldwide. These technologies have allowed for the rapid analysis, to date, of the genomes of more than 2,000 different species. In humans, NGS has arguably had the largest impact. Over 100,000 genomes of individual humans (based on various estimates have been sequenced allowing for deep insights into what makes individuals and families unique and what causes disease in each of us. Despite all of this progress, the current state of the art in sequence technology is far from generating a perfect genome sequence and much remains to be understood in the biology of human and other organisms’ genomes. In the article that follows we outline, why the perfect genome in humans is important, what is lacking from current human whole genome sequences, and a potential strategy for achieving the perfect genome in a cost effective manner.

  5. Fluorescence studies on the interaction of ethidium bromide with duplex, triplex and quadruplex DNA structures

    Institute of Scientific and Technical Information of China (English)

    孙雪光; 曹恩华; 何裕建; 秦静芬

    1999-01-01

    Under different conditions, oligonucleotides can form several alternative DNA structures such as duplex, triplex and quadruplex. All these structures can interact with ethidium bromide (EB) and make its fluorescence intensity change. The fluorescence spectra and other related parameters provided by static fluorescence techniques showed that the interaction mechanisms between EB and these structures were not always the same. Among them, B type duplex and triplex DNA adopt an intercalative mode when binding to the EB, which has a relatively high efficiency of energy transfer and the fluorescence of EB cannot be quenched easily. While for the parallel duplex DNA, the interaction mode is an outside binding in which energy transfer can hardly happen and its fluorescence intensity as well as Stern-Volmer constant is almost the same to the free EB. For the quadruplex, the binding mechanism to EB is more complex. Results from the energy transfer and quenching studies indicate that the two interaction modes note

  6. Direct Fluorescent Detection of Blood Potassium by Ion-Selective Formation of Intermolecular G-Quadruplex and Ligand Binding.

    Science.gov (United States)

    Yang, Le; Qing, Zhihe; Liu, Changhui; Tang, Qiao; Li, Jishan; Yang, Sheng; Zheng, Jing; Yang, Ronghua; Tan, Weihong

    2016-09-20

    G-quadruplex analogues have been widely used as molecular tools for detection of potassium ion (K(+)). However, interference from a higher concentration of sodium ion (Na(+)), enzymatic degradation of the oligonucleotide, and background absorption and fluorescence of blood samples have all limited the use of G-quadruplex for direct detection of K(+) in blood samples. Here, we reported, for the first time, an intermolecular G-quadruplex-based assay capable of direct fluorescent detection of blood K(+). Increased stringency of intermolecular G-quadruplex formation based on our screened G-rich oligonucleotide (5'-TGAGGGA GGGG-3') provided the necessary selectivity for K(+) against Na(+) at physiological ion level. To increase long-term stability of oligonucleotide in blood, the screened oligonucleotide was modified with an inverted thymine nucleotide whose 3'-terminus was connected to the 3'-terminus of the upstream nucleotide, acting as a blocking group to greatly improve antinuclease stability. Lastly, to avoid interference from background absorption and autofluorescence of blood, a G-quadruplex-binding, two-photon-excited ligand, EBMVC-B, was synthesized and chosen as the fluorescence reporter. Thus, based on selective K(+) ion-induced formation of intermolecular G-quadruplex and EBMVC-B binding, this approach could linearly respond to K(+) from 0.5 to 10 mM, which matches quite well with the physiologically relevant concentration of blood K(+). Moreover, the system was highly selective for K(+) against other metal ions, including Na(+), Ca(2+), Mg(2+), Zn(2+) common in blood. The practical application was demonstrated by direct detection of K(+) from real blood samples by two-photon fluorescence technology. To the best of our knowledge, this is the first attempt to exploit molecular G-quadruplex-based fluorescent sensing for direct assay of blood target. As such, we expect that it will promote the design and practical application of similar DNA-based sensors in

  7. Macrocyclic hexaoxazoles: Influence of aminoalkyl substituents on RNA and DNA G-quadruplex stabilization and cytotoxicity.

    Science.gov (United States)

    Satyanarayana, Mavurapu; Kim, Young-Ah; Rzuczek, Suzanne G; Pilch, Daniel S; Liu, Angela A; Liu, Leroy F; Rice, Joseph E; LaVoie, Edmond J

    2010-05-15

    A series of 24-membered macrocyclic hexaoxazoles containing one or two aminoalkyl substituents was synthesized and evaluated for cytotoxicity and for their ability to selectively stabilize G-quadruplex DNA and RNA. The most cytotoxic analog 4a, with IC(50) values of 25 and 130 nM using KB3-1 and RPMI 8402 cells, is efficacious in vivo in athymic nude mice with a human tumor xenograft from the breast cancer cell line MDA-MB-435.

  8. Native Electrospray Mass Spectrometry of DNA G-Quadruplexes in Potassium Solution

    OpenAIRE

    2014-01-01

    A commonly used electrolyte in electrospray mass spectrometry (ESI-MS) of biomolecules is ammonium acetate (NH4OAc). Although some nucleic acid structures such as duplexes require only proper physiological ionic strength (whatever the monovalent ions) to be properly folded in ESI-MS conditions, the folding of some other nucleic acid structures such as DNA G-quadruplexes also depends on direct binding of specific cations. Here, we developed ESI-MS compatible conditions that allow one to observ...

  9. Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process.

    Science.gov (United States)

    Reshetnikov, Roman V; Sponer, Jiri; Rassokhina, Olga I; Kopylov, Alexei M; Tsvetkov, Philipp O; Makarov, Alexander A; Golovin, Andrey V

    2011-12-01

    A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange.

  10. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes.

    Science.gov (United States)

    Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

    2015-12-02

    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation.

  11. Identification of a New G-Quadruplex Motif in the KRAS Promoter and Design of Pyrene-Modified G4-Decoys with Antiproliferative Activity in Pancreatic Cancer Cells

    DEFF Research Database (Denmark)

    Cogoi, Susanna; Paramasivam, Manikandan; Filitchev, Vyacheslav Viatcheslav;

    2009-01-01

    A new quadruplex motif located in the promoter of the human KRAS gene, within a nuclease hypersensitive element (NHE), has been characterized. Oligonucleotides mimicking this quadruplex are found to compete with a DNA-protein complex between NHE and a nuclear extract from pancreatic cancer cells...

  12. A streptavidin paramagnetic-particle based competition assay for the evaluation of the optical selectivity of quadruplex nucleic acid fluorescent probes.

    Science.gov (United States)

    Largy, Eric; Hamon, Florian; Teulade-Fichou, Marie-Paule

    2012-05-01

    Although quadruplex nucleic acids are thought to be involved in many biological processes, they are massively overwhelmed by duplex DNA in the cell. Small molecules, able to probe quadruplex nucleic acids with high optical selectivity, could possibly achieve the visualization of these processes. The aim of the method described herein is to evaluate quickly the optical selectivity of quadruplex nucleic acid probes, in isothermal conditions, using widely available materials, small quantities of oligonucleotides and virtually any kind and quantity of biological competitor. The assay relies on the use of streptavidin-coated paramagnetic particles and biotinylated quadruplex forming oligonucleotides, allowing a quick and easy separation of the quadruplex target from the competitor. In the present study, two quadruplex nucleic acids (the DNA and RNA human telomeric repeats) have been used as targets while a duplex DNA oligonucleotide, total DNA, total RNA, another quadruplex nucleic acid and a protein have been used as competitors. The optical selectivity of various probes, displaying different photophysical properties and binding selectivities, has been successfully examined, allowing the identification of a best candidate for further cell microscopy experiments. This assay allows a quick and reliable assessment of the labeling properties of a quadruplex binder in cellular environment conditions. It is an interesting alternative to gel electrophoresis experiments since it is performed in solution, has a well-resolved separation system and allows easy quantifications.

  13. EFFECT OF DYE CONCENTRATION ON SEQUENCING BATCH REACTOR PERFORMANCE

    Directory of Open Access Journals (Sweden)

    A. A. Vaigan ، M. R. Alavi Moghaddam ، H. Hashemi

    2009-01-01

    Full Text Available Reactive dyes have been identified as problematic compounds in textile industries wastewater as they are water soluble and cannot be easily removed by conventional aerobic biological treatment systems. The treatability of a reactive dye (Brill Blue KN-R by sequencing batch reactor and the influence of the dye concentration on system performance were investigated in this study. Brill Blue KN-R is one of the main dyes that are used in textile industries in Iran. Four cylindrical Plexiglas reactors were run for 36 days (5 days for acclimatization of sludge and 31 days for normal operation at different initial dye concentrations. The dye concentrations were adjusted to be 20, 25, 30 and 40 mg/L in the reactors R1, R2, R3 and R4, respectively. In all reactors, effective volume, influent wastewater flowrate and sludge retention time were 5.5 L, 3.0 L/d and 10 d, respectively. According to the obtained data, average dye removal efficiencies of R1, R2, R3 and R4 were 57% ± 2, 50.18% ± 3, 44.97% ± 3 and 30.98% ± 3, respectively. The average COD removal efficiencies of all reactors were 97% ± 1, 97.12% ± 1, 96.93% ± 1 and 97.22% ± 1, respectively. The dye removal efficiency was decreased by increasing the dye concentration with the correlation coefficient of 0.997.

  14. Topology of a G-quadruplex DNA formed by C9orf72 hexanucleotide repeats associated with ALS and FTD.

    Science.gov (United States)

    Zhou, Bo; Liu, Changdong; Geng, Yanyan; Zhu, Guang

    2015-11-13

    Abnormal expansions of an intronic hexanucleotide GGGGCC (G4C2) repeat of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies suggested that the C9orf72 hexanucleotide repeat expansion (HRE), either as DNA or the transcribed RNA, can fold into G-quadruplexes with distinct structures. These structural polymorphisms lead to abortive transcripts and contribute to the pathogenesis of ALS and FTD. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, we analyzed the structures of C9orf72 HRE DNA with various G4C2 repeats. They exhibited diverse G-quadruplex folds in potassium ions. Furthermore, we determined the topology of a G-quadruplex formed by d(G4C2)4. It favors a monomeric fold and forms a chair-type G-quadruplex with a four-layer antiparallel G-tetra core and three edgewise loops, which is distinct from known structures of chair-type G-quadruplexes. Our findings highlight the conformational heterogeneity of C9orf72 HRE DNA, and may lay the necessary structural basis for designing small molecules for the modulation of ALS/FTD pathogenesis.

  15. A new application of click chemistry in situ: development of fluorescent probe for specific G-quadruplex topology.

    Science.gov (United States)

    Hu, Ming-Hao; Chen, Xiao; Chen, Shuo-Bin; Ou, Tian-Miao; Yao, Meicun; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2015-11-25

    Target-guided synthesis is an approach to drug discovery that allows the target to self-assemble its own binding agents. So far, target-guided synthesis and especially in situ click chemistry have attracted extensive attention and have led to the identification of highly potent inhibitors for proteins. In this study, we expand the application of in situ click chemistry and present a procedure using this approach to identify selective fluorescent probes for a specific topology of G-quadruplex nucleic acids, the parallel G-quadruplexes. On this basis, compound 15 assembled by triarylimidazole scaffold and carboxyl side chain was a positive hit, demonstrating highly potential in the sensitive and selective detection of parallel G-quadruplexes. Such selective fluorescence response can be rationalized in terms of different binding affinities between 15 and G-quadruplexes. Our work accordingly represents a new development towards the application of in situ click chemistry to develop selective fluorescent probes and may also shed light on the search for probes for a specific G-quadruplex topology.

  16. Effects of Sequence on Transmission Properties of DNA Molecules

    Institute of Scientific and Technical Information of China (English)

    DONG Rui-Xin; YAN Xun-Ling; YANG Bing

    2008-01-01

    A double helix model of charge transport in DNA molecule is given and the transmission spectra of four DNA sequences are obtained. The calculated results show that the transmission characteristics of DNA are not only related to the longitudinal transport but also to the transverse transport of molecule. The periodic sequence with the same composition has stronger conduction ability. With the increasing of bases composition, the conductive ability reduces, but the weight of θ direction rises in charge transfer.

  17. Correlated mutations in protein sequences: Phylogenetic and structural effects

    Energy Technology Data Exchange (ETDEWEB)

    Lapedes, A.S. [Los Alamos National Lab., NM (United States). Theoretical Div.]|[Santa Fe Inst., NM (United States); Giraud, B.G. [C.E.N. Saclay, Gif/Yvette (France). Service Physique Theorique; Liu, L.C. [Los Alamos National Lab., NM (United States). Theoretical Div.; Stormo, G.D. [Univ. of Colorado, Boulder, CO (United States). Dept. of Molecular, Cellular and Developmental Biology

    1998-12-01

    Covariation analysis of sets of aligned sequences for RNA molecules is relatively successful in elucidating RNA secondary structure, as well as some aspects of tertiary structure. Covariation analysis of sets of aligned sequences for protein molecules is successful in certain instances in elucidating certain structural and functional links, but in general, pairs of sites displaying highly covarying mutations in protein sequences do not necessarily correspond to sites that are spatially close in the protein structure. In this paper the authors identify two reasons why naive use of covariation analysis for protein sequences fails to reliably indicate sequence positions that are spatially proximate. The first reason involves the bias introduced in calculation of covariation measures due to the fact that biological sequences are generally related by a non-trivial phylogenetic tree. The authors present a null-model approach to solve this problem. The second reason involves linked chains of covariation which can result in pairs of sites displaying significant covariation even though they are not spatially proximate. They present a maximum entropy solution to this classic problem of causation versus correlation. The methodologies are validated in simulation.

  18. Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU.

    Directory of Open Access Journals (Sweden)

    Emmanuel O Ariyo

    Full Text Available Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes.

  19. Effects of KLK Peptide on Adjuvanticity of Different ODN Sequences

    Directory of Open Access Journals (Sweden)

    Ghania Chikh

    2016-05-01

    Full Text Available Endosomal Toll-like receptors (TLR such as TLR3, 7, 8 and 9 recognize pathogen associated nucleic acids. While DNA sequence does influence degree of binding to and activation of TLR9, it also appears to influence the ability of the ligand to reach the intracellular endosomal compartment. The KLK (KLKL5KLK antimicrobial peptide, which is immunostimulatory itself, can translocate into cells without cell membrane permeabilization and thus can be used for endosomal delivery of TLR agonists, as has been shown with the IC31 formulation that contains an oligodeoxynucleotide (ODN TLR9 agonist. We evaluated the adjuvant activity of KLK combined with CpG or non-CpG (GpC ODN synthesized with nuclease resistant phosphorothioate (S or native phosphodiester (O backbones with ovalbumin (OVA antigen in mice. As single adjuvants, CpG(S gave the strongest enhancement of OVA-specific immunity and the addition of KLK provided no benefit and was actually detrimental for some readouts. In contrast, KLK enhanced the adjuvant effects of CpG(O and to a lesser extent of GpC (S, which on their own had little or no activity. Indeed while CD8 T cells, IFN-γ secretion and humoral response to vaccine antigen were enhanced when CpG(O was combined with KLK, only IFN-γ secretion was enhanced when GpC (S was combined to KLK. The synergistic adjuvant effects with KLK/ODN combinations were TLR9-mediated since they did not occur in TLR9 knock-out mice. We hypothesize that a nuclease resistant ODN with CpG motifs has its own mechanism for entering cells to reach the endosome. For ODN without CpG motifs, KLK appears to provide an alternate mechanism for accessing the endosome, where it can activate TLR9, albeit with lower potency than a CpG ODN. For nuclease sensitive (O backbone ODN, KLK may also provide protection from nucleases in the tissues.

  20. G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold

    DEFF Research Database (Denmark)

    Marusic, Maja; Veedu, Rakesh N; Wengel, Jesper

    2013-01-01

    The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes...... residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G...... exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue...

  1. Effects of Early Musical Experience on Auditory Sequence Memory.

    Science.gov (United States)

    Tierney, Adam T; Bergeson-Dana, Tonya R; Pisoni, David B

    2008-10-01

    The present study investigated a possible link between musical training and immediate memory span by testing experienced musicians and three groups of musically inexperienced subjects (gymnasts, Psychology 101 students, and video game players) on sequence memory and word familiarity tasks. By including skilled gymnasts who began studying their craft by age six, video game players, and Psychology 101 students as comparison groups, we attempted to control for some of the ways skilled musicians may differ from participants drawn from the general population in terms of gross motor skills and intensive experience in a highly skilled domain from an early age. We found that musicians displayed longer immediate memory spans than the comparison groups on auditory presentation conditions of the sequence reproductive span task. No differences were observed between the four groups on the visual conditions of the sequence memory task. These results provide additional converging support to recent findings showing that early musical experience and activity-dependent learning may selectively affect verbal rehearsal processes and the allocation of attention in sequence memory tasks.

  2. Effects of Early Musical Experience on Auditory Sequence Memory

    Directory of Open Access Journals (Sweden)

    Adam T. Tierney

    2008-12-01

    Full Text Available The present study investigated a possible link between musical training and immediate memory span by testing experienced musicians and three groups of musically inexperienced subjects (gymnasts, Psychology 101 students, and video game players on sequence memory and word familiarity tasks. By including skilled gymnasts who began studying their craft by age six, video game players, and Psychology 101 students as comparison groups, we attempted to control for some of the ways skilled musicians may differ from participants drawn from the general population in terms of gross motor skills and intensive experience in a highly skilled domain from an early age. We found that musicians displayed longer immediate memory spans than the comparison groups on auditory presentation conditions of the sequence reproductive span task. No differences were observed between the four groups on the visual conditions of the sequence memory task. These results provide additional converging support to recent findings showing that early musical experience and activity-dependent learning may selectively affect verbal rehearsal processes and the allocation of attention in sequence memory tasks.

  3. Circular dichroism spectroscopic investigation of double-decker phthalocyanine with G-Quadruplex as promising telomerase inhibitor

    Science.gov (United States)

    Baǧda, Efkan; Baǧda, Esra; Yabaş, Ebru

    2017-01-01

    In the present study, interaction of a double-decker phthalocyanine with two G-quadruplex DNA, Tel 21 and cMYC, was investigated. To the best of our knowledge, this is the first study about G-quadruplex-double decker phthalocyanine interaction. The spectrophotometric titration method was used for binding constant calculations. From the binding constants, it can be said that double-decker phthalocyanine more likely to bind Tel 21 rather than cMYC. The conformational changes upon binding were monitored via circular dichroism spectroscopy. The ethidium bromide replacement assay was investigated spectrofluorometrically.

  4. Effective noninvasive zygosity determination by maternal plasma target region sequencing.

    Directory of Open Access Journals (Sweden)

    Jing Zheng

    Full Text Available BACKGROUND: Currently very few noninvasive molecular genetic approaches are available to determine zygosity for twin pregnancies in clinical laboratories. This study aimed to develop a novel method to determine zygosity by using maternal plasma target region sequencing. METHODS: We constructed a statistic model to calculate the possibility of each zygosity type using likelihood ratios ( Li and empirical dynamic thresholds targeting at 4,524 single nucleotide polymorphisms (SNPs loci on 22 autosomes. Then two dizygotic (DZ twin pregnancies,two monozygotic (MZ twin pregnancies and two singletons were recruited to evaluate the performance of our novel method. Finally we estimated the sensitivity and specificity of the model in silico under different cell-free fetal DNA (cff-DNA concentration and sequence depth. RESULTS/CONCLUSIONS: We obtained 8.90 Gbp sequencing data on average for six clinical samples. Two samples were classified as DZ with L values of 1.891 and 1.554, higher than the dynamic DZ cut-off values of 1.162 and 1.172, respectively. Another two samples were judged as MZ with 0.763 and 0.784 of L values, lower than the MZ cut-off values of 0.903 and 0.918. And the rest two singleton samples were regarded as MZ twins, with L values of 0.639 and 0.757, lower than the MZ cut-off values of 0.921 and 0.799. In silico, the estimated sensitivity of our noninvasive zygosity determination was 99.90% under 10% total cff-DNA concentration with 2 Gbp sequence data. As the cff-DNA concentration increased to 15%, the specificity was as high as 97% with 3.50 Gbp sequence data, much higher than 80% with 10% cff-DNA concentration. SIGNIFICANCE: This study presents the feasibility to noninvasively determine zygosity of twin pregnancy using target region sequencing, and illustrates the sensitivity and specificity under various detecting condition. Our method can act as an alternative approach for zygosity determination of twin pregnancies in clinical

  5. Effective DNA fragmentation technique for simple sequence repeat detection with a microsatellite-enriched library and high-throughput sequencing.

    Science.gov (United States)

    Tanaka, Keisuke; Ohtake, Rumi; Yoshida, Saki; Shinohara, Takashi

    2017-04-01

    Two different techniques for genomic DNA fragmentation before microsatellite-enriched library construction-restriction enzyme (NlaIII and MseI) digestion and sonication-were compared to examine their effects on simple sequence repeat (SSR) detection using high-throughput sequencing. Tens of thousands of SSR regions from 5 species of the plant family Myrtaceae were detected when the output of individual samples was >1 million paired-end reads. Comparison of the two DNA fragmentation techniques showed that restriction enzyme digestion was superior to sonication for identification of heterozygous genotypes, whereas sonication was superior for detection of various SSR flanking regions with both species-specific and common characteristics. Therefore, choosing the most suitable DNA fragmentation method depends on the type of analysis that is planned.

  6. The Effects of Explicit Instruction of Formulaic Sequences on Second-Language Writers

    Science.gov (United States)

    Colovic-Markovic, Jelena

    2012-01-01

    The present study investigated the effects of the explicit teaching of formulaic sequences (i.e., academic and topic-induced) on L2 writing. The research examined separately the effects of the treatment on the students' abilities to produce the target formulaic sequences in controlled (i.e., C-tests) and uncontrolled situations (i.e.,…

  7. Base Sequence Context Effects on Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    Yuqin Cai

    2010-01-01

    Full Text Available Nucleotide excision repair (NER plays a critical role in maintaining the integrity of the genome when damaged by bulky DNA lesions, since inefficient repair can cause mutations and human diseases notably cancer. The structural properties of DNA lesions that determine their relative susceptibilities to NER are therefore of great interest. As a model system, we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene (B[a]P, 10S (+-trans-anti-B[a]P-2-dG in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups. We have obtained molecular structural data by NMR and MD simulations, bending properties from gel electrophoresis studies, and NER data obtained from human HeLa cell extracts for our six investigated sequence contexts. This model system suggests that disturbed Watson-Crick base pairing is a better recognition signal than a flexible bend, and that these can act in concert to provide an enhanced signal. Steric hinderance between the minor groove-aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances. Both nearest neighbor and more distant neighbor sequence contexts have an impact. Regardless of the exact distortions, we hypothesize that they provide a local thermodynamic destabilization signal for repair.

  8. Sequence complexity effects on speech production in healthy speakers and speakers with hypokinetic or ataxic dysarthria.

    Science.gov (United States)

    Reilly, Kevin J; Spencer, Kristie A

    2013-01-01

    The present study investigated the effects of sequence complexity, defined in terms of phonemic similarity and phonotoactic probability, on the timing and accuracy of serial ordering for speech production in healthy speakers and speakers with either hypokinetic or ataxic dysarthria. Sequences were comprised of strings of consonant-vowel (CV) syllables with each syllable containing the same vowel, /a/, paired with a different consonant. High complexity sequences contained phonemically similar consonants, and sounds and syllables that had low phonotactic probabilities; low complexity sequences contained phonemically dissimilar consonants and high probability sounds and syllables. Sequence complexity effects were evaluated by analyzing speech error rates and within-syllable vowel and pause durations. This analysis revealed that speech error rates were significantly higher and speech duration measures were significantly longer during production of high complexity sequences than during production of low complexity sequences. Although speakers with dysarthria produced longer overall speech durations than healthy speakers, the effects of sequence complexity on error rates and speech durations were comparable across all groups. These findings indicate that the duration and accuracy of processes for selecting items in a speech sequence is influenced by their phonemic similarity and/or phonotactic probability. Moreover, this robust complexity effect is present even in speakers with damage to subcortical circuits involved in serial control for speech.

  9. Sequence complexity effects on speech production in healthy speakers and speakers with hypokinetic or ataxic dysarthria.

    Directory of Open Access Journals (Sweden)

    Kevin J Reilly

    Full Text Available The present study investigated the effects of sequence complexity, defined in terms of phonemic similarity and phonotoactic probability, on the timing and accuracy of serial ordering for speech production in healthy speakers and speakers with either hypokinetic or ataxic dysarthria. Sequences were comprised of strings of consonant-vowel (CV syllables with each syllable containing the same vowel, /a/, paired with a different consonant. High complexity sequences contained phonemically similar consonants, and sounds and syllables that had low phonotactic probabilities; low complexity sequences contained phonemically dissimilar consonants and high probability sounds and syllables. Sequence complexity effects were evaluated by analyzing speech error rates and within-syllable vowel and pause durations. This analysis revealed that speech error rates were significantly higher and speech duration measures were significantly longer during production of high complexity sequences than during production of low complexity sequences. Although speakers with dysarthria produced longer overall speech durations than healthy speakers, the effects of sequence complexity on error rates and speech durations were comparable across all groups. These findings indicate that the duration and accuracy of processes for selecting items in a speech sequence is influenced by their phonemic similarity and/or phonotactic probability. Moreover, this robust complexity effect is present even in speakers with damage to subcortical circuits involved in serial control for speech.

  10. The Effect of Pre-Main Sequence Stars on Star Cluster Dynamics

    CERN Document Server

    Wiersma, R; Zwart, S P

    2006-01-01

    We investigate the effects of the addition of pre-main sequence evolution to star cluster simulations. We allowed stars to follow pre-main sequence tracks that begin at the deuterium burning birthline and end at the zero age main sequence. We compared our simulations to ones in which the stars began their lives at the zero age main sequence, and also investigated the effects of particular choices for initial binary orbital parameters. We find that the inclusion of the pre-main sequence phase results in a slightly higher core concentration, lower binary fraction, and fewer hard binary systems. In general, the global properties of star clusters remain almost unchanged, but the properties of the binary star population in the cluster can be dramatically modified by the correct treatment of the pre-main sequence stage.

  11. Novel FeII and CoII Complexes of Natural Product Tryptanthrin: Synthesis and Binding with G-Quadruplex DNA

    Science.gov (United States)

    Zhong, Yi-ning; Zhang, Yan; Gu, Yun-qiong; Wu, Shi-yun; Shen, Wen-ying

    2016-01-01

    Tryptanthrin is one of the most important members of indoloquinoline alkaloids. We obtained this alkaloid from Isatis. Two novel FeII and CoII complexes of tryptanthrin were first synthesized. Single-crystal X-ray diffraction analyses show that these complexes display distorted four-coordinated tetrahedron geometry via two heterocyclic nitrogen and oxygen atoms from tryptanthrin ligand. Binding with G-quadruplex DNA properties revealed that both complexes were found to exhibit significant interaction with G-quadruplex DNA. This study may potentially serve as the basis of future rational design of metal-based drugs from natural products that target the G-quadruplex DNA. PMID:27698647

  12. Target guided synthesis using DNA nano-templates for selectively assembling a G-quadruplex binding c-MYC inhibitor

    Science.gov (United States)

    Panda, Deepanjan; Saha, Puja; Das, Tania; Dash, Jyotirmayee

    2017-07-01

    The development of small molecules is essential to modulate the cellular functions of biological targets in living system. Target Guided Synthesis (TGS) approaches have been used for the identification of potent small molecules for biological targets. We herein demonstrate an innovative example of TGS using DNA nano-templates that promote Huisgen cycloaddition from an array of azide and alkyne fragments. A G-quadruplex and a control duplex DNA nano-template have been prepared by assembling the DNA structures on gold-coated magnetic nanoparticles. The DNA nano-templates facilitate the regioselective formation of 1,4-substituted triazole products, which are easily isolated by magnetic decantation. The G-quadruplex nano-template can be easily recovered and reused for five reaction cycles. The major triazole product, generated by the G-quadruplex inhibits c-MYC expression by directly targeting the c-MYC promoter G-quadruplex. This work highlights that the nano-TGS approach may serve as a valuable strategy to generate target-selective ligands for drug discovery.

  13. Computational understanding and experimental characterization of twice-as-smart quadruplex ligands as chemical sensors of bacterial nucleotide second messengers

    Science.gov (United States)

    Zhou, Jie; Roembke, Benjamin T.; Paragi, Gabor; Laguerre, Aurélien; Sintim, Herman O.; Fonseca Guerra, Célia; Monchaud, David

    2016-01-01

    A twice-as-smart ligand is a small molecule that experiences a structural switch upon interaction with its target (i.e., smart ligand) that concomitantly triggers its fluorescence (i.e., smart probe). Prototypes of twice-as-smart ligands were recently developed to track and label G-quadruplexes: these higher-order nucleic acid structures originate in the assembly of four guanine(G)-rich DNA or RNA strands, whose stability is imparted by the formation and the self-assembly of G-quartets. The first prototypes of twice-as-smart quadruplex ligands were designed to exploit the self-association of quartets, being themselves synthetic G-quartets. While their quadruplex recognition capability has been thoroughly documented, some doubts remain about the precise photophysical mechanism that underlies their peculiar spectroscopic properties. Here, we uncovered this mechanism via complete theoretical calculations. Collected information was then used to develop a novel application of twice-as-smart ligands, as efficient chemical sensors of bacterial signaling pathways via the fluorescent detection of naturally occurring extracellular quadruplexes formed by cyclic dimeric guanosine monophosphate (c-di-GMP). PMID:27667717

  14. Polymeric membrane neutral phenol-sensitive electrodes for potentiometric G-quadruplex/hemin DNAzyme-based biosensing.

    Science.gov (United States)

    Wang, Xuewei; Ding, Zhaofeng; Ren, Qingwei; Qin, Wei

    2013-02-05

    The first potentiometric transducer for G-quadruplex/hemin DNAzyme-based biosensing has been developed by using potential responses of electrically neutral oligomeric phenols on polymeric membrane electrodes. In the presence of G-quadruplex/hemin DNAzyme and H(2)O(2), monomeric phenols (e.g., phenol, methylphenols, and methoxyphenols) can be condensed into oligomeric phenols. Because both substrates and products are nonionic under optimal pH conditions, these reactions are traditionally not considered in designing potentiometric biosensing schemes. However, in this paper, the electrically neutral oligomeric phenols have been found to induce highly sensitive potential responses on quaternary ammonium salt-doped polymeric membrane electrodes owing to their high lipophilicities. In contrast, the potential responses to monomeric phenolic substrates are rather low. Thus, the G-quadruplex/hemin DNAzyme-catalyzed oxidative coupling of monomeric phenols can induce large potential signals, and the catalytic activities of DNAzymes can be probed. A comparison of potential responses induced by peroxidations of 13 monomeric phenols indicates that p-methoxyphenol is the most efficient substrate for potentiometric detection of G-quadruplex/hemin DNAzymes. Finally, two label-free and separation-free potentiometric DNA assay protocols based on the G-quadruplex/hemin DNAzyme have been developed with sensitivities higher than those of colorimetric and fluorometric methods. Coupled with other features such as reliable instrumentation, low cost, ease of miniaturization, and resistance to color and turbid interferences, the proposed polymeric membrane-based potentiometric sensor promises to be a competitive transducer for peroxidase-mimicking DNAzyme-involved biosensing.

  15. Biological activity of the G-quadruplex ligand RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate) is associated with telomere capping alteration.

    Science.gov (United States)

    Leonetti, Carlo; Amodei, Sarah; D'Angelo, Carmen; Rizzo, Angela; Benassi, Barbara; Antonelli, Anna; Elli, Raffaella; Stevens, Malcolm F G; D'Incalci, Maurizio; Zupi, Gabriella; Biroccio, Annamaria

    2004-11-01

    This study had two goals: 1) to evaluate the biological effect of the novel pentacyclic acridine 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) on human melanoma lines possessing long telomeres, and 2) to elucidate the relationship between G-quadruplex-based telomerase inhibitor-induced cellular effects and telomere length/dysfunction. The cellular pharmacological effects of RHPS4 have been evaluated by treating melanoma lines with increasing concentrations of RHPS4. A dose-dependent inhibition of cell proliferation was observed in all the lines during short-term treatment. Flow cytometric analysis demonstrated that RHPS4 induced a dose-dependent accumulation of cells in the S-G(2)/M phase of cell cycle. The RHPS4-induced cell cycle alteration was irreversible even at low doses, and the cells died from apoptosis. At high RHPS4 concentration, apoptosis was accompanied by the induction of a senescence phenotype: large cell size, vacuolated cytoplasm, and beta-galactosidase activity. The short-term biological activity of RHPS4 was not caused by telomere shortening, but it was associated with telomere dysfunction, in terms of presence of telomeric fusions, polynucleated cells, and typical images of telophase bridge. In conclusion, our results demonstrate that the G-quadruplex ligand RHPS4 can function in a telomere length-independent manner through its ability to cause telomere-capping alteration.

  16. “One Ring to Bind Them All”—Part I: The Efficiency of the Macrocyclic Scaffold for G-Quadruplex DNA Recognition

    Directory of Open Access Journals (Sweden)

    David Monchaud

    2010-01-01

    Full Text Available Macrocyclic scaffolds are particularly attractive for designing selective G-quadruplex ligands essentially because, on one hand, they show a poor affinity for the “standard” B-DNA conformation and, on the other hand, they fit nicely with the external G-quartets of quadruplexes. Stimulated by the pioneering studies on the cationic porphyrin TMPyP4 and the natural product telomestatin, follow-up studies have developed, rapidly leading to a large diversity of macrocyclic structures with remarkable-quadruplex binding properties and biological activities. In this review we summarize the current state of the art in detailing the three main categories of quadruplex-binding macrocycles described so far (telomestatin-like polyheteroarenes, porphyrins and derivatives, polyammonium cyclophanes, and in addressing both synthetic issues and biological aspects.

  17. The effect of task difficulty on eye movement sequences in multiple dimensions

    NARCIS (Netherlands)

    Dewhurst, Richard; Nyström, Marcus; Jarodzka, Halszka; Foulsham, Tom; Johansson, Roger; Holmqvist, Kenneth

    2012-01-01

    Dewhurst, R., Nyström, M., Jarodzka, H., Foulsham, T., Johansson, R., & Holmqvist, K. (2012, May). The effect of task difficulty on eye movement sequences in multiple dimensions. Presentation at the Scandinavian Workshop on Applied Eye Tracking, Stockholm, Sweden.

  18. effect of sequences of ozone and nitrogen dioxide on plant dry ...

    African Journals Online (AJOL)

    Prof. Adipala Ekwamu

    implication of sequences of these two gases is very important. Hence, the ... effect on growth after 21 days; while NO2 caused a significant increase in growth only when applied alone early. Exposures to NO2 .... A greenhouse experiment was.

  19. “Turn-off-on” fluorescent sensor for (N-methyl-4-pyridyl) porphyrin -DNA and G-quadruplex interactions based on ZnCdSe quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Dan; Fan, Yao; Gao, Fang; Yang, Tian-ming, E-mail: tmyang@mail.scuec.edu.cn

    2015-08-12

    As a new detection model, the reversible fluorescence “turn-off-on” sensor based on quantum dots (QDs) has already been successfully employed in the detections of many biochemical materials, especially in the researches on the interactions between anticancer drugs. The previous studies, however, mainly focused on simple-structured oligonucleotides and Calf thymus DNA. G-quadruplex, an important target for anti-cancer drug with special secondary structure, has been stimulating increasing research interests. In this paper, we report a new detection method based on the fluorescence “turn-off-on” model with water-soluble ZnCdSe QDs as the fluorescent probe, to analyze the interactions between anticancer drug (N-methyl-4-pyridyl) porphyrin (TMPyP) and nucleic acid, especially the G-quadruplex. The fluorescence of QDs can be quenched by TMPyP via photo-induced electron transfer and fluorescence resonance energy transfer, while on the other hand, the combination between TMPyP and G-quadruplex releases QDs from their quenchers and thus recovers the fluorescence. Most importantly, the fluorescence “turn-off-on” model has been employed, for the first time, to analyze the impacts of special factors on the interaction between TMPyP and G-quadruplex. The excellent selectivity of the system has been verified in the studies of the interactions between TMPyP and different DNAs (double-stranded DNA, single-stranded G-quadruplex, and different types of G-quadruplexes) in Na{sup +} or K{sup +}-containing buffer. - Highlights: • Reversible fluorescence sensor was firstly used on TMPyP and G-quadruplex study. • SsDNA and various G-quadruplexes were successfully recognized by fluorescence. • The new quantum dot is hypotoxicity and can be extensively applied.

  20. Modelling studies on neurodegenerative disease-causing triplet repeat sequences d(GGC/GCC)n and d(CAG/CTG)n

    Indian Academy of Sciences (India)

    Shibasish Chowdhury; Manju Bansal

    2001-12-01

    Model building and molecular mechanics studies have been carried out to examine the potential structures for d(GGC/GCC)5 and d(CAG/CTG)5 that might relate to their biological function and association with triplet repeat expansion diseases. Model building studies suggested that hairpin and quadruplex structures could be formed with these repeat sequences. Molecular mechanics studies have demonstrated that the hairpin and hairpin dimer structures of triplet repeat sequences formed by looping out of the two strands are as favourable as the corresponding B-DNA type hetero duplex structures. Further, at high salt condition, Greek key type quadruplex structures are energetically comparable with hairpin dimer and B-DNA type duplex structures. All tetrads in the quadruplex structures are well stacked and provide favourable stacking energy values. Interestingly, in the energy minimized hairpin dimer and Greek key type quadruplex structures, all the bases even in the non-G tetrads are cyclically hydrogen bonded, even though the A, C and T-tetrads were not hydrogen bonded in the starting structures.

  1. NMR structure of dual site binding of mitoxantrone dimer to opposite grooves of parallel stranded G-quadruplex [d-(TTGGGGT)]4.

    Science.gov (United States)

    Pradeep, Tarikere Palakshan; Barthwal, Ritu

    2016-01-01

    The formation of complex between anti-cancer drug mitoxantrone (MTX) and tetra-molecular parallel G-quadruplex DNA [d-(TTGGGGT)]4 has been studied by solution state one and two dimensional NMR spectroscopy. Mitoxantrone forms a head-to-tail dimer and binds at two opposite grooves of the G-quadruplex. The Job's method of continuous variation and thermal melting studies independently ascertain binding stoichiometry of 4:1 in mitoxantrone:DNA complex. The existence of only four guanine NH peaks corresponding to the four G-quartets during the course of titration shows that C4 symmetry of G-quadruplex is intact upon binding of mitoxantrone. The specific inter molecular short distance contacts between protons of two mitoxantrone molecules of dimer, that is, ring A protons with ring C and side chain methylene protons, confirms the formation of mitoxantrone head-to-tail dimer. The observed 38 Nuclear Overhauser Enhancement (NOE) cross peaks between MTX and G-quadruplex DNA indicate formation of a well-defined complex. The three dimensional structure of 4:1 mitoxantrone:[d-(TTGGGGT)]4 complex computed by using experimental distance restraints followed by restrained Molecular Dynamics (rMD) simulations envisages the critical knowledge of specific molecular interactions within ligand-G-quadruplex complex. The findings are of direct interest in development of anti-cancer therapeutic drug based on G-quadruplex stabilization, resulting in telomerase inhibition.

  2. Phylo-typing of clinical Escherichia coli isolates originating from bovine mastitis and canine pyometra and urinary tract infection by means of quadruplex PCR.

    Science.gov (United States)

    Müştak, Hamit Kaan; Günaydin, Elçin; Kaya, İnci Başak; Salar, Merve Özdal; Babacan, Orkun; Önat, Kaan; Ata, Zafer; Diker, Kadir Serdar

    2015-01-01

    Escherichia coli is one of the major causative agents of bovine mastitis worldwide, and is typically associated with acute, clinical mastitis. Besides this, E. coli strains which belong to the extra-intestinal pathogenic group are also the major cause of urinary tract infections and pyometra in dogs. In this study, it was aimed to investigate phylo-groups/subgroups in 155 E. coli isolates obtained from acute bovine mastitis, 43 from urinary tract infections of dogs and 20 from canine pyometra by a formerly described triplex PCR and recently described new quadruplex polymerase chain reaction (PCR) method. Group A1 (n = 118; 76%) and B1 (n = 71; 46%) were found to be the most prevalent groups by triplex and quadruplex PCR assays in mastitis isolates, respectively. Phylo-typing of 43 urinary tract isolates also revealed that most of the isolates belonged to A1 (n = 23; 54%) by triplex and B2 (n = 36; 84%) by quadruplex PCR assays. The isolates assigned as group A1 (n = 17; 85%) by triplex PCR could not be classified by quadruplex PCR in pyometra isolates. The results support the hypothesis that E. coli strains isolated from bovine mastitis cases are environmental. Also, groups C, E and F were identified as new phylo-groups for the first time in acute bovine mastitis cases. The comparison of triplex PCR with quadruplex PCR results revealed that most of the groups assigned in triplex PCR were altered by quadruplex PCR assay.

  3. Examining the Effect of Task Complexity and Sequence on Speaking Ability of Iranian EFL Learners

    Directory of Open Access Journals (Sweden)

    Masoumeh Alipour Madarsara

    2015-01-01

    Full Text Available The impetus of the present study was to examine the effect of task complexity and sequence on speaking according to performance data collected from 60 intermediate Iranian EFL learners on two tasks (a map task and a car task. In order to examine the effects of task sequence and complexity in enhancing EFL learners’ speaking ability in three different areas including accuracy, fluency and complexity, descriptive statistics as well as independent samples T-tests were run to the results of each sections of the speaking test for both control and experimental groups in posttest. It was found that task sequence and complexity had significant effects on Iranian intermediate EFL learners’ speaking ability. The findings of the study also revealed that the participants in the experimental group, who practiced task sequence and complexity, far outweighed the control group in complexity and fluency than the other area of the speaking test.

  4. A Cost-Effective Approach to Sequence Hundreds of Complete Mitochondrial Genomes.

    Science.gov (United States)

    Nunez, Joaquin C B; Oleksiak, Marjorie F

    2016-01-01

    We present a cost-effective approach to sequence whole mitochondrial genomes for hundreds of individuals. Our approach uses small reaction volumes and unmodified (non-phosphorylated) barcoded adaptors to minimize reagent costs. We demonstrate our approach by sequencing 383 Fundulus sp. mitochondrial genomes (192 F. heteroclitus and 191 F. majalis). Prior to sequencing, we amplified the mitochondrial genomes using 4-5 custom-made, overlapping primer pairs, and sequencing was performed on an Illumina HiSeq 2500 platform. After removing low quality and short sequences, 2.9 million and 2.8 million reads were generated for F. heteroclitus and F. majalis respectively. Individual genomes were assembled for each species by mapping barcoded reads to a reference genome. For F. majalis, the reference genome was built de novo. On average, individual consensus sequences had high coverage: 61-fold for F. heteroclitus and 57-fold for F. majalis. The approach discussed in this paper is optimized for sequencing mitochondrial genomes on an Illumina platform. However, with the proper modifications, this approach could be easily applied to other small genomes and sequencing platforms.

  5. Effects of priming goal pursuit on implicit sequence learning

    OpenAIRE

    Gamble, Katherine R.; Lee, Joanna M.; Howard, James H.; Howard, Darlene V.

    2014-01-01

    Implicit learning, the type of learning that occurs without intent to learn or awareness of what has been learned, has been thought to be insensitive to the effects of priming, but recent studies suggest this is not the case. One study found that learning in the Serial Reaction Time (SRT) task was improved by nonconscious goal pursuit, primed via a word search task (Eitam et al., 2008). In two studies, we used the goal priming word search task from Eitam et al., but with a different version o...

  6. Structural Dynamics of Thrombin-Binding DNA Aptamer d(GGTTGGTGTGGTTGG) Quadruplex DNA Studied by Large-Scale Explicit Solvent Simulations.

    Science.gov (United States)

    Reshetnikov, Roman; Golovin, Andrey; Spiridonova, Vera; Kopylov, Alexei; Šponer, Jiří

    2010-10-12

    The thrombin-binding aptamer (15-TBA) is a 15-mer DNA oligonucleotide with sequence d(GGTTGGTGTGGTTGG). 15-TBA folds into a quadruplex DNA (G-DNA) structure with two planar G-quartets connected by three single-stranded loops. The arrangement of the 15-TBA-thrombin complex is unclear, particularly with respect to the precise 15-TBA residues that interact with the thrombin structure. Our present understanding suggests either the 15-TBA single stranded loops containing sequential thymidines (TT) or alternatively a single-stranded loop, containing a guanine flanked by 2 thymidines (TGT), physically associates with thrombin protein. In the present study, the explicit solvent molecular dynamics (MD) simulation method was utilized to further analyze the 15-TBA-thrombin three-dimensional structure. Functional annotation of the loop residues was made with long simulations in the parmbsc0 force field. In total, the elapsed time of simulations carried out in this study exceeds 12 microseconds, substantially surpassing previous G-DNA simulation reports. Our simulations suggest that the TGT-loop function is to stabilize the structure of the aptamer, while the TT-loops participate in direct binding to thrombin. The findings of the present report advance our understanding of the molecular structure of the 15-TBA-thrombin structure further enabling the construction of biosensors for aptamer bases and the development of anticoagulant agents.

  7. Two valuation questions in one survey: Is it a recipe for sequencing and instrument context effects?

    Science.gov (United States)

    Giraud, K.L.; Loomis, J.B.; Johnson, R.L.

    1999-01-01

    Economic theory suggests that willingness to pay for two goods independently offered should remain unchanged when the survey instrument changes slightly. Four survey treatments consisting of comprehensive good and a subset of that good were used. The surveys alternated in the question ordering and in the embedded good which accompanied the comprehensive good. We tested for sequencing and instrument context effects using both a combined and split sample designs. In the combined sample case we found some evidence to sequencing effects in the data containing the first subset good. Likelihood ratio tests indicated that sequencing did not effect scale or location of parameters. In the test for instrument context effects, evidence was found indicating context does effect willingness to pay estimates.

  8. Sequence effect in Parkinson’s disease is related to motor energetic cost

    Directory of Open Access Journals (Sweden)

    Sule eTinaz

    2016-05-01

    Full Text Available Bradykinesia is the most disabling motor symptom of Parkinson’s disease (PD. The sequence effect, a feature of bradykinesia, refers to the rapid decrement in amplitude and speed of repetitive movements (e.g., gait, handwriting and is a major cause of morbidity in PD. Previous research has revealed mixed results regarding the role of dopaminergic treatment in the sequence effect. However, external cueing has been shown to improve it. In this study, we aimed to characterize the sequence effect systematically and relate this phenomenon to the energetic cost of movement within the context of cost-benefit framework of motor control. We used a dynamic isometric motor task with auditory pacing to assess the sequence effect in motor output during a 15 s task segment in PD patients and matched controls. All participants performed the task with both hands, and without and with visual feedback. Patients were also tested in on- and off-dopaminergic states. Patients in the off state did not show higher sequence effect compared to controls, partly due to large variance in their performance. However, patients in the on state and in the absence of visual feedback, showed significantly higher sequence effect compared to controls. Patients expended higher total motor energy compared to controls in all conditions and regardless of their medication status. In this experimental situation, the sequence effect in PD is associated with the cumulative energetic cost of movement. Dopaminergic treatment, critical for internal triggering of movement, fails to maintain the motor vigor across responses. The high motor cost may be related to failure to incorporate limbic/motivational cues into the motor plan. Visual feedback may facilitate performance by shifting the driving of movement from internal to external, or, alternatively, by functioning as a motivational cue.

  9. Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs.

    Science.gov (United States)

    Yang, Jun-Bo; Li, De-Zhu; Li, Hong-Tao

    2014-09-01

    Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle-scale barcodes. Next-generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high-quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long-range PCR and sequenced using next-generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early-diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome-scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms. © 2014 John Wiley & Sons Ltd.

  10. Heterogeneous Suppression of Sequential Effects in Random Sequence Generation, but Not in Operant Learning

    Science.gov (United States)

    Shteingart, Hanan; Loewenstein, Yonatan

    2016-01-01

    There is a long history of experiments in which participants are instructed to generate a long sequence of binary random numbers. The scope of this line of research has shifted over the years from identifying the basic psychological principles and/or the heuristics that lead to deviations from randomness, to one of predicting future choices. In this paper, we used generalized linear regression and the framework of Reinforcement Learning in order to address both points. In particular, we used logistic regression analysis in order to characterize the temporal sequence of participants’ choices. Surprisingly, a population analysis indicated that the contribution of the most recent trial has only a weak effect on behavior, compared to more preceding trials, a result that seems irreconcilable with standard sequential effects that decay monotonously with the delay. However, when considering each participant separately, we found that the magnitudes of the sequential effect are a monotonous decreasing function of the delay, yet these individual sequential effects are largely averaged out in a population analysis because of heterogeneity. The substantial behavioral heterogeneity in this task is further demonstrated quantitatively by considering the predictive power of the model. We show that a heterogeneous model of sequential dependencies captures the structure available in random sequence generation. Finally, we show that the results of the logistic regression analysis can be interpreted in the framework of reinforcement learning, allowing us to compare the sequential effects in the random sequence generation task to those in an operant learning task. We show that in contrast to the random sequence generation task, sequential effects in operant learning are far more homogenous across the population. These results suggest that in the random sequence generation task, different participants adopt different cognitive strategies to suppress sequential dependencies when

  11. Prosodic effects on glide-vowel sequences in three Romance languages

    Science.gov (United States)

    Chitoran, Ioana

    2004-05-01

    Glide-vowel sequences occur in many Romance languages. In some they can vary in production, ranging from diphthongal pronunciation [ja,je] to hiatus [ia,ie]. According to native speakers' impressionistic perceptions, Spanish and Romanian both exhibit this variation, but to different degrees. Spanish favors glide-vowel sequences, while Romanian favors hiatus, occasionally resulting in different pronunciations of the same items: Spanish (b[j]ela, ind[j]ana), Romanian (b[i]ela, ind[i]ana). The third language, French, has glide-vowel sequences consistently (b[j]elle). This study tests the effect of position in the word on the acoustic duration of the sequences. Shorter duration indicates diphthong production [jV], while longer duration, hiatus [iV]. Eleven speakers (4 Spanish, 4 Romanian, 3 French), were recorded. Spanish and Romanian showed a word position effect. Word-initial sequences were significantly longer than word-medial ones (p0.05). In the Spanish and Romanian sentences, V in the sequence bears pitch accent, but not in French. It is therefore possible that duration is sensitive not to the presence/absence of the word boundary, but to its position relative to pitch accent. The results suggest that the word position effect is crucially enhanced by pitch accent on V.

  12. Long-range charge transport in single G-quadruplex DNA molecules

    DEFF Research Database (Denmark)

    Livshits, Gideon I.; Stern, Avigail; Rotem, Dvir

    2014-01-01

    -ups, and transporting significant current through individual DNA-based molecules remains a considerable challenge. Here, we report reproducible charge transport in guanine-quadruplex (G4) DNA molecules adsorbed on a mica substrate. Currents ranging from tens of picoamperes to more than 100 pA were measured in the G4......-DNA over distances ranging from tens of nanometres to more than 100 nm. Our experimental results, combined with theoretical modelling, suggest that transport occurs via a thermally activated long-range hopping between multi-tetrad segments of DNA. These results could re-ignite interest in DNA......DNA and DNA-based polymers are of interest in molecular electronics because of their versatile and programmable structures. However, transport measurements have produced a range of seemingly contradictory results due to differences in the measured molecules and experimental set...

  13. A quadruplex PCR (qxPCR) assay for adulteration in dairy products.

    Science.gov (United States)

    Agrimonti, Caterina; Pirondini, Andrea; Marmiroli, Marta; Marmiroli, Nelson

    2015-11-15

    This study describes the development of a quadruplex quantitative Real Time PCR (qxPCR) based on SYBR®GreenER chemistry, for rapid identification of DNA of cow, goat, sheep and buffalo in dairy products, and for quantification of cow DNA in these products. The platform was applied to: (i) mixes of milks at fixed percentages; (ii) cheeses prepared with the same mixes; (iii) commercial dairy products. The methodology enabled the detection of DNA from cow in mixes of milk and cheeses with a limit of detection (LOD) of 0.1%. When applied to commercial dairy products the qxPCR gave results comparable with each single-plex Real Time PCR. A good correlation (R(2)>0.9) between peaks' area of derivative of melting curves of amplicons and percentages of cow milk in milk mixes and cheeses, allows for an estimation of cow DNA in a dynamic range varying from 0.1-5% to 1-25%.

  14. The effect of attentional load on implicit sequence learning in children and young adults

    Directory of Open Access Journals (Sweden)

    Daphné eCoomans

    2014-05-01

    Full Text Available We investigated the effect of a secondary task on implicit sequence learning in children and young adults. A serial reaction time task was administered to 8-to-10 year old children and 18-to-22 year old adults. Participants reacted to the location of a target presented in one of four locations on the screen with a spatially corresponding response key. Unknown to participants, the location at which the target appeared was structured according to a deterministic sequence. Occasionally, the black target dot was replaced by a red target dog. To assess the effect of attentional load on implicit sequence learning, half of the participants of each age group was assigned to the single task condition, while the other half executed the task under dual task conditions. Whereas participants in the single task condition could ignore the change in target identity, dual task participants additionally had to count the number of times the black dot was replaced by a red dog to increase the attentional load. Sequence learning was tested under single task conditions in both conditions. Z-transformed results indicate that young adults generally showed more sequence learning than children. Importantly, the secondary task had no effect on sequence learning in children, since children learned as much under dual task conditions as under single task conditions. Adults, on the other hand, showed a different result pattern, as they displayed more sequence learning under single task than under dual task conditions. We surmise that this result is due to the vainly attempt of adults, but not children, to integrate both sequences.

  15. Diffusion measurements for molecular capsules: pulse sequences effect on water signal decay.

    Science.gov (United States)

    Avram, Liat; Cohen, Yoram

    2005-04-20

    Diffusion NMR and, more recently, diffusion ordered spectroscopy (DOSY) are gaining popularity as efficient tools for the characterization of supramolecular systems in solution. Here, using diffusion NMR of hydrogen-bond molecular capsules, we demonstrate that the use of different diffusion sequences may have a dramatic effect on exchanging peaks. In fact, we found that the signal decay of the water peak in [(1a)(6)(H(2)O)(8)] is monoexponential in the pulsed gradient spin-echo (PGSE) and stimulated echo (PGSTE) sequences and biexponential in the longitudinal eddy current delay (LED) and the bipolar longitudinal eddy current delay (BPLED) sequences, routinely used in modern DOSY experiments. By performing these diffusion measurements on molecular capsules, in which water is not part of the molecular capsules, we demonstrate that this phenomenon is observed only for water molecules that exchange between two sites that differ considerably in their diffusion coefficients. Degeneration of the LED or the BPLED sequences into PGSTE-type sequences by shortening the te period resulted in the disappearance of the extra slow diffusing component. The origin, as well as the implications of the different results obtained from conventional diffusion sequences, such as the PGSE and PGSTE as compared with the LED and BPLED sequences generally used in DOSY experiments, are briefly discussed.

  16. Effects of interlinker sequences on the biological properties of bispecific single-chain antibodies

    Institute of Scientific and Technical Information of China (English)

    FANG Min; JIANG Xin; YANG Zhi; YIN Changcheng; LI Hua; ZHAO Rui; ZHANG Zhong; LIN Qing; HUANG Hualiang

    2003-01-01

    Single-chain bispecific antibody (scBsAb) is one of the promising genetic engineering antibody formats for clinical application. But the effects of interlinker sequences on the biological properties of bispecific single-chain antibodies have not been studied in detail. Three interlinker sequences were designed and synthesized, and denominated as Fc, HSA, 205C′, respectively. Universal vectors with these different interlinker sequences for scBsAb expression in E. coli were constructed. A model scBsAb based on a reshaped single-chain antibody (scFv) against human CD3 and a scFv directed against human ovarian carcinoma were generated and expressed in E. coli. The results of SDS-PAGE and Western blot showed that the different interlinker sequences did not affect the expression levelof scBsAb. However, as demonstrated by ELISA and pharmacokinetics studies performed in mice, scBsAbs with different interlinker sequences had difference in the antigen-binding activities and terminal half-life time (T1/2β) in vivo, the interlinker HSA could remarkably prolong the retention time of scBsAb in blood. These results indicated that the peptide sequence of interlinker could affect important biological properties of scBsAb, such as antigen-binding properties and stability in vivo. So, selection of an appropriate interlinker sequence is very important for scBsAb construction. Optimal interlinker can bring scBsAb biologicalproperties more suitable for clinical application.

  17. Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing.

    Science.gov (United States)

    Campbell, Nathan R; Harmon, Stephanie A; Narum, Shawn R

    2015-07-01

    Genotyping-in-Thousands by sequencing (GT-seq) is a method that uses next-generation sequencing of multiplexed PCR products to generate genotypes from relatively small panels (50-500) of targeted single-nucleotide polymorphisms (SNPs) for thousands of individuals in a single Illumina HiSeq lane. This method uses only unlabelled oligos and PCR master mix in two thermal cycling steps for amplification of targeted SNP loci. During this process, sequencing adapters and dual barcode sequence tags are incorporated into the amplicons enabling thousands of individuals to be pooled into a single sequencing library. Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences. Genotyping is performed with a simple perl script which counts amplicon-specific sequences for each allele, and allele ratios are used to determine the genotypes. We demonstrate this technique by genotyping 2068 individual steelhead trout (Oncorhynchus mykiss) samples with a set of 192 SNP markers in a single library sequenced in a single Illumina HiSeq lane. Genotype data were 99.9% concordant to previously collected TaqMan(™) genotypes at the same 192 loci, but call rates were slightly lower with GT-seq (96.4%) relative to Taqman (99.0%). Of the 192 SNPs, 187 were genotyped in ≥90% of the individual samples and only 3 SNPs were genotyped in <70% of samples. This study demonstrates amplicon sequencing with GT-seq greatly reduces the cost of genotyping hundreds of targeted SNPs relative to existing methods by utilizing a simple library preparation method and massive efficiency of scale.

  18. Including load sequence effects in the fatigue damage estimation of an offshore wind turbine substructure

    NARCIS (Netherlands)

    Dragt, R.C.; Maljaars, J.; Tuitman, J.T.; Salman, Y.; Otheguy, M.E.

    2016-01-01

    Retardation is a load sequence effect, which causes a reduced fatigue crack growth rate after an overload is encountered. Retardation can be cancelled when the overload is followed by an underload. The net effect is beneficial to the fatigue lifetime of Offshore Wind Turbines (OWTs). To be able to t

  19. Polypeptoids: A model system for exploring sequence and shape effects on block copolymer self-assembly

    Science.gov (United States)

    Segalman, Rachel

    2015-03-01

    While our ability to understand the detailed relationship between block copolymer chemistry and mesoscopic self-assembly has made remarkable progress over the last many years, yet we are still limited to a relatively small number of blocks in terms of structure-property understanding. Thus, there is a need to explore self-assembly phase space with sequence using a model system. Polypeptoids are non-natural, sequence specific polymers that offer the opportunity to probe the effect of sequence on self-assembly with much simpler molecular interactions and more scalable synthesis than traditional polypeptides. In this talk, I will discuss the use of this model system to understand the role of sequence on chain collapse and globule formation in solution, polymer crystallization, and block copolymer self-assembly. I will then discuss potential application as surface active agents for anti-fouling.

  20. Partial sequence and toxic effects of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

    Directory of Open Access Journals (Sweden)

    A.N.C. Santana

    1998-10-01

    Full Text Available A neurotoxic peptide, granulitoxin (GRX, was isolated from the sea anemone Bunodosoma granulifera. The N-terminal amino acid sequence of GRX is AKTGILDSDGPTVAGNSLSGT and its molecular mass is 4958 Da by electrospray mass spectrometry. This sequence presents a partial degree of homology with other toxins from sea anemones such as Bunodosoma caissarum, Anthopleura fuscoviridis and Anemonia sulcata. However, important differences were found: the first six amino acids of the sequence are different, Arg-14 was replaced by Ala and no cysteine residues were present in the partial sequence, while two cysteine residues were present in the first 21 amino acids of other toxins described above. Purified GRX injected ip (800 µg/kg into mice produced severe neurotoxic effects such as circular movements, aggressive behavior, dyspnea, tonic-clonic convulsion and death. The 2-h LD50 of GRX was 400 ± 83 µg/kg.

  1. Effective automated feature construction and selection for classification of biological sequences.

    Directory of Open Access Journals (Sweden)

    Uday Kamath

    Full Text Available Many open problems in bioinformatics involve elucidating underlying functional signals in biological sequences. DNA sequences, in particular, are characterized by rich architectures in which functional signals are increasingly found to combine local and distal interactions at the nucleotide level. Problems of interest include detection of regulatory regions, splice sites, exons, hypersensitive sites, and more. These problems naturally lend themselves to formulation as classification problems in machine learning. When classification is based on features extracted from the sequences under investigation, success is critically dependent on the chosen set of features.We present an algorithmic framework (EFFECT for automated detection of functional signals in biological sequences. We focus here on classification problems involving DNA sequences which state-of-the-art work in machine learning shows to be challenging and involve complex combinations of local and distal features. EFFECT uses a two-stage process to first construct a set of candidate sequence-based features and then select a most effective subset for the classification task at hand. Both stages make heavy use of evolutionary algorithms to efficiently guide the search towards informative features capable of discriminating between sequences that contain a particular functional signal and those that do not.To demonstrate its generality, EFFECT is applied to three separate problems of importance in DNA research: the recognition of hypersensitive sites, splice sites, and ALU sites. Comparisons with state-of-the-art algorithms show that the framework is both general and powerful. In addition, a detailed analysis of the constructed features shows that they contain valuable biological information about DNA architecture, allowing biologists and other researchers to directly inspect the features and potentially use the insights obtained to assist wet-laboratory studies on retainment or modification

  2. 2'-F-ANA-guanosine and 2'-F-guanosine as powerful tools for structural manipulation of G-quadruplexes.

    Science.gov (United States)

    Lech, Christopher Jacques; Li, Zhe; Heddi, Brahim; Phan, Anh Tuân

    2012-12-04

    Here we demonstrate the applicability of 2'-F-ANA-guanosine and 2'-F-guanosine as powerful tools for manipulating G-quadruplex folding by anti-position-favoring substitutions. A single guanine to 2'-F-ANA-guanine substitution can favor a single (3+1) hybrid conformation from a mixture of conformers. Rational substitutions of either type of 2'-F-modified nucleotide enable conformational switching from a (3+1) hybrid to a parallel folding topology.

  3. Ring-closing metathesis for the synthesis of a highly G-quadruplex selective macrocyclic hexaoxazole having enhanced cytotoxic potency.

    Science.gov (United States)

    Satyanarayana, Mavurapu; Rzuczek, Suzanne G; Lavoie, Edmond J; Pilch, Daniel S; Liu, Angela; Liu, Leroy F; Rice, Joseph E

    2008-07-01

    The synthesis of a 24-membered macrocyclic hexaoxazole via ring-closing metathesis is described. The target compound selectively stabilizes G-quadruplex DNA with no detectable stabilization of duplex DNA. An MTT cytotoxicity assay indicated that this unsaturated macrocyclic hexaoxazole exhibits significant cytotoxicity toward P388, RPMI 8402, and KB3-1 cell lines with IC50 values of 45, 25, and 38 nM, respectively.

  4. CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours

    Science.gov (United States)

    Xu, Hong; Di Antonio, Marco; McKinney, Steven; Mathew, Veena; Ho, Brandon; O'Neil, Nigel J.; Santos, Nancy Dos; Silvester, Jennifer; Wei, Vivien; Garcia, Jessica; Kabeer, Farhia; Lai, Daniel; Soriano, Priscilla; Banáth, Judit; Chiu, Derek S.; Yap, Damian; Le, Daniel D.; Ye, Frank B.; Zhang, Anni; Thu, Kelsie; Soong, John; Lin, Shu-chuan; Tsai, Angela Hsin Chin; Osako, Tomo; Algara, Teresa; Saunders, Darren N.; Wong, Jason; Xian, Jian; Bally, Marcel B.; Brenton, James D.; Brown, Grant W.; Shah, Sohrab P.; Cescon, David; Mak, Tak W.; Caldas, Carlos; Stirling, Peter C.; Hieter, Phil; Balasubramanian, Shankar; Aparicio, Samuel

    2017-01-01

    G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016). PMID:28211448

  5. Action-effect congruence during observational learning leads to faster action sequence learning.

    Science.gov (United States)

    Horvath, Jared C; Gray, Zachary; Schilberg, Lukas; Vidrin, Ilya; Pascual-Leone, Alvaro

    2015-01-01

    Common coding theory suggests that any action (pressing a piano key) is intimately linked with its resultant sensory effect (an auditory musical tone). We conducted two experiments to explore the effect of varying auditory action-effect patterns during complex action learning. In Experiment 1, participants were assigned to 1 of 4 groups, watched a silent video of a hand playing a sequence on a piano keyboard with no auditory action effect (observation) and were asked to practise and perform the sequence on an identical keyboard with varying action effects (reproduction). During reproduction, Group 1 heard no auditory tones (identical to observed video), Group 2 heard typical scale-ascending piano tones with each key press, Group 3 heard fixed but out-of-sequence piano tones with each key press, and Group 4 heard random piano tones with each key press. In Experiment two, new participants were assigned to 1 of 2 groups and watched an identical video; however, the video in this experiment contained typical, scale-ascending piano sounds. During reproduction, Group 1 heard no auditory tones while Group 2 heard typical, scale-ascending piano tones with each key press (identical to observed video). Our results showed that participants whose action-effect patterns during reproduction matched those in the observed video learned the action sequence faster than participants whose action-effect patterns during reproduction differed from those in the observed video. Additionally, our results suggest that adding an effect during reproduction (when one is absent during observation) is somewhat more detrimental to action sequence learning than removing an effect during reproduction (when one is present during observation).

  6. Effect of multimedia information sequencing on educational outcome in orthodontic training.

    Science.gov (United States)

    Aly, Medhat; Willems, Guy; Van Den Noortgate, Wim; Elen, Jan

    2012-08-01

    The aim of this research was to compare the effectiveness of hierarchical sequencing (HS) versus elaboration sequencing (ES) models in improving educational outcome of clinical knowledge when using instructional multimedia programs in postgraduate orthodontic training. Twenty-four postgraduate and 24 undergraduate dental students participated in this study. The postgraduates were following an orthodontic speciality training programme. The undergraduates were fourth- and fifth-year dental students. Twelve instructional multimedia modules were developed, six logically sequenced (LS) discussing six different orthodontic topics. Another six modules on identical topics were sequenced according to one macro-sequencing (MS) model. The implemented MS model was either HS or ES. The only difference between LS and MS modules was the adopted sequencing model. All participants were assigned into consistent pairs of students and were randomly divided into a test and a control group. In each pair, one student studied the LS module (control group) while the other studied the MS version (test group). Pre- and post-evaluation tests of each pair of participants were performed to measure knowledge, understanding and application of each participant with regard to the discussed topic. A multilevel analysis was conducted to assess the estimated effect of the different sequencing models. The level of significance was set at 0.05. At baseline, no significant differences (P > 0.05) were found in pre-test scores between groups. The HS model showed a significant effect on the scores achieved (P = 0.05). The test group showed a significantly higher estimated probability of correct answers to the questions (P = 0.003) when applying the HS model. The HS model may improve educational outcome when using instructional multimedia programs in postgraduate orthodontic training.

  7. Strong position-dependent effects of sequence mismatches on signal ratios measured using long oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Hulme Helen

    2008-07-01

    Full Text Available Abstract Background Microarrays are an important and widely used tool. Applications include capturing genomic DNA for high-throughput sequencing in addition to the traditional monitoring of gene expression and identifying DNA copy number variations. Sequence mismatches between probe and target strands are known to affect the stability of the probe-target duplex, and hence the strength of the observed signals from microarrays. Results We describe a large-scale investigation of microarray hybridisations to murine probes with known sequence mismatches, demonstrating that the effect of mismatches is strongly position-dependent and for small numbers of sequence mismatches is correlated with the maximum length of perfectly matched probe-target duplex. Length of perfect match explained 43% of the variance in log2 signal ratios between probes with one and two mismatches. The correlation with maximum length of perfect match does not conform to expectations based on considering the effect of mismatches purely in terms of reducing the binding energy. However, it can be explained qualitatively by considering the entropic contribution to duplex stability from configurations of differing perfect match length. Conclusion The results of this study have implications in terms of array design and analysis. They highlight the significant effect that short sequence mismatches can have upon microarray hybridisation intensities even for long oligonucleotide probes. All microarray data presented in this study are available from the GEO database 1, under accession number [GEO: GSE9669

  8. Consolidating the effects of waking and sleep on motor-sequence learning.

    Science.gov (United States)

    Brawn, Timothy P; Fenn, Kimberly M; Nusbaum, Howard C; Margoliash, Daniel

    2010-10-20

    Sleep is widely believed to play a critical role in memory consolidation. Sleep-dependent consolidation has been studied extensively in humans using an explicit motor-sequence learning paradigm. In this task, performance has been reported to remain stable across wakefulness and improve significantly after sleep, making motor-sequence learning the definitive example of sleep-dependent enhancement. Recent work, however, has shown that enhancement disappears when the task is modified to reduce task-related inhibition that develops over a training session, thus questioning whether sleep actively consolidates motor learning. Here we use the same motor-sequence task to demonstrate sleep-dependent consolidation for motor-sequence learning and explain the discrepancies in results across studies. We show that when training begins in the morning, motor-sequence performance deteriorates across wakefulness and recovers after sleep, whereas performance remains stable across both sleep and subsequent waking with evening training. This pattern of results challenges an influential model of memory consolidation defined by a time-dependent stabilization phase and a sleep-dependent enhancement phase. Moreover, the present results support a new account of the behavioral effects of waking and sleep on explicit motor-sequence learning that is consistent across a wide range of tasks. These observations indicate that current theories of memory consolidation that have been formulated to explain sleep-dependent performance enhancements are insufficient to explain the range of behavioral changes associated with sleep.

  9. Effects of the bleaching sequence on the optical brighteners action in eucalyptus kraft pulp

    Directory of Open Access Journals (Sweden)

    Mauro Manfredi

    2014-06-01

    Full Text Available During the bleaching process the pulp is treated with chemical reagents that can be retained in the pulp and interfere in the action of the optical brighteners. Different bleaching sequences can produce pulps at the same brightness but with different potential to whiteness increase when treated with optical brighteners. The objective of this study was to evaluate the influence of the bleaching sequence on the efficiency of disulphonated and tetrasulphonated optical brighteners. Eucalyptus kraft pulp was bleached using four different bleaching sequences. For each pulp three brightness targets were aimeds. For each bleaching sequence mathematical model was generated for predicting the final pulp whiteness according to the initial brightness and the optical brightener charge applied. The presence of organochlorine residues in the pulp reduced the effectiveness of the optical brighteners. Therefore, bleaching sequences that use low chlorine dioxide charge favors for greater gains in whiteness with the application of optical brighteners. The replacement of the final chlorine dioxide bleaching stage with a hydrogen peroxide one in the sequence increased the efficiency of the optical brightening agents.

  10. A Study on the Effect of Welding Sequence in Fabrication of Large Stiffened Plate Panels

    Institute of Scientific and Technical Information of China (English)

    Pankaj Biswas; D.Anil Kumar; N.R.Mandal; M.M.Mahapatra

    2011-01-01

    Welding sequence has a significant effect on distortion pattern of large orthogonally stiffened panels normally used in ships and offshore structures.These deformations adversely affect the subsequent fitup and alignment of the adjacent panels.It may also result in loss of structural integrity.These panels primarily suffer from angular and buckling distortions.The extent of distortion depends on several parameters such as welding speed,plate thickness,welding current,voltage,restraints applied to the job while welding,thermal history as well as sequence of welding.Numerical modeling of welding and experimental validation of the FE model has been carried out for estimation of thermal history and resulting distortions.In the present work an FE model has been developed for studying the effect of welding sequence on the distortion pattern and its magnitude in fabrication of orthogonally stiffened plate panels.

  11. Structural varieties of selectively mixed G- and C-rich short DNA sequences studied with electrospray ionization mass spectrometry.

    Science.gov (United States)

    Cao, Yanwei; Gao, Shang; Li, Caijin; Yan, Yuting; Wang, Bing; Guo, Xinhua

    2016-10-01

    Short guanine(G)-repeat and cytosine(C)-repeat DNA strands can self-assemble to form four-stranded G-quadruplexes and i-motifs, respectively. Herein, G-rich and C-rich strands with non-G or non-C terminal bases and different lengths of G- or C-repeats are mixed selectively in pH 4.5 and 6.7 ammonium acetate buffer solutions and studied by electrospray ionization mass spectrometry (ESI-MS). Various strand associations corresponding to bi-, tri- and tetramolecular ions are observed in mass spectra, indicating that the formation of quadruplex structures is a random strand by strand association process. However, with increasing incubation time for the mixtures, initially associated hybrid tetramers will transform into self-assembled conformations, which is mainly driven by the structural stability. The melting temperature values of self-assembled quadruplexes suggest that the length of G-repeats or C-repeats shows more significant effect on the stability of quadruplex structures than that of terminal residues. Accordingly, we can obtain the self-associated tetrameric species generated from the mixtures of various homologous G- or C-strands efficiently by altering the length of G- or C-repeats. Our studies demonstrate that ESI-MS is a very direct, fast and sensitive tool to provide significant information on DNA strand associations and stoichiometric transitions, particularly for complex mixtures. Copyright © 2016 John Wiley & Sons, Ltd.

  12. Effects of aging and dopamine genotypes on the emergence of explicit memory during sequence learning.

    Science.gov (United States)

    Schuck, Nicolas W; Frensch, Peter A; Schjeide, Brit-Maren M; Schröder, Julia; Bertram, Lars; Li, Shu-Chen

    2013-11-01

    The striatum and medial temporal lobe play important roles in implicit and explicit memory, respectively. Furthermore, recent studies have linked striatal dopamine modulation to both implicit as well as explicit sequence learning and suggested a potential role of the striatum in the emergence of explicit memory during sequence learning. With respect to aging, previous findings indicated that implicit memory is less impaired than explicit memory in older adults and that genetic effects on cognition are magnified by aging. To understand the links between these findings, we investigated effects of aging and genotypes relevant for striatal dopamine on the implicit and explicit components of sequence learning. Reaction time (RT) and error data from 80 younger (20-30 years) and 70 older adults (60-71 years) during a serial reaction time task showed that age differences in learning-related reduction of RTs emerged gradually over the course of learning. Verbal recall and measures derived from the process-dissociation procedure revealed that younger adults acquired more explicit memory about the sequence than older adults, potentially causing age differences in RT gains in later stages of learning. Of specific interest, polymorphisms of the dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32, rs907094) and dopamine transporter (DAT, VNTR) genes showed interactive effects on overall RTs and verbal recall of the sequence in older but not in younger adults. Together our findings show that variations in genotypes relevant for dopamine functions are associated more with aging-related impairments in the explicit than the implicit component of sequence learning, providing support for theories emphasizing the role of dopaminergic modulation in cognitive aging and the magnification of genetic effects in human aging. © 2013 Elsevier Ltd. All rights reserved.

  13. Electrochemical aptasensor based on the dual-amplification of G-quadruplex horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish peroxidase.

    Science.gov (United States)

    Yuan, Yali; Gou, Xuxu; Yuan, Ruo; Chai, Yaqin; Zhuo, Ying; Mao, Li; Gan, Xianxue

    2011-06-15

    A simple electrochemical aptasensor for sensitive detection of thrombin was fabricated with G-quadruplex horseradish peroxidase-mimicking DNAzyme (hemin/G-quadruplex system) and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. Gold nanoparticles (nano-Au) were firstly electrodeposited onto single wall nanotube (SWNT)-graphene modified electrode surface for the immobilization of electrochemical probe of nickel hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was electrodeposited for further immobilization of thrombin aptamer (TBA), which later formed hemin/G-quadruplex system with hemin. Horseradish peroxidases (HRP) then served as blocking reagent to block possible remaining active sites and avoided the non-specific adsorption. In the presence of thrombin, the TBA binded to thrombin and the hemin released from the hemin/G-quadruplex electrocatalytic structure, increasing steric hindrance of the aptasensor and decomposing hemin/G-quadruplex electrocatalytic structure, which finally decreased the electrocatalytic efficiency of aptasensor toward H(2)O(2) in the presence of NiHCFNPs with a decreased electrochemical signal. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained.

  14. Ultrafast Electron Transfer in Complexes of Doxorubicin with Human Telomeric G-Quadruplexes and GC Duplexes Probed by Femtosecond Fluorescence Spectroscopy.

    Science.gov (United States)

    Changenet-Barret, Pascale; Gustavsson, Thomas; Markovitsi, Dimitra; Manet, Ilse

    2016-05-04

    Doxorubicin (DOX) is a natural anthracycline widely used in chemotherapy; its combined application as a chemotherapeutic and photodynamic agent has been recently proposed. In this context, understanding the photoinduced properties of DOX complexes with nucleic acids is crucial. Herein, the study of photoinduced electron transfer in DOX-DNA complexes by femtosecond fluorescence spectroscopy is reported. The behaviour of complexes with two model DNA structures, a G-quadruplex (G4) formed by the human telomeric sequence (Tel21) and a d(GC) duplex, is compared. The DOX affinity for these two sequences is similar. Although both 1:1 and 2:1 stoichiometries have been reported for DOX-G4 complexes, only 1:1 complexes form with the duplex. The steady-state absorption indicates a strong binding interaction with the duplex due to drug intercalation between the GC base pairs. In contrast, the interaction of DOX with Tel21 is much weaker and arises from drug binding on the G4 external faces at two independent binding sites. As observed for DOX-d(GC) complexes, fluorescence of the drug in the first binding site of Tel21 exhibits decays within a few picoseconds following a biphasic pattern; this is attributed to the existence of two drug conformations. The fluorescence of the drug in the second binding site of Tel21 shows slower decays within 150 ps. These timescales are consistent with electron transfer from the guanines to the excited drug, as favoured by the lower oxidation potential of the stacked guanines of G4 with respect to those in the duplex.

  15. Thermoelectric effect and its dependence on molecular length and sequence in single DNA molecules.

    Science.gov (United States)

    Li, Yueqi; Xiang, Limin; Palma, Julio L; Asai, Yoshihiro; Tao, Nongjian

    2016-01-01

    Studying the thermoelectric effect in DNA is important for unravelling charge transport mechanisms and for developing relevant applications of DNA molecules. Here we report a study of the thermoelectric effect in single DNA molecules. By varying the molecular length and sequence, we tune the charge transport in DNA to either a hopping- or tunnelling-dominated regimes. The thermoelectric effect is small and insensitive to the molecular length in the hopping regime. In contrast, the thermoelectric effect is large and sensitive to the length in the tunnelling regime. These findings indicate that one may control the thermoelectric effect in DNA by varying its sequence and length. We describe the experimental results in terms of hopping and tunnelling charge transport models.

  16. Picture or Text First? Explaining Sequence Effects When Learning with Pictures and Text

    Science.gov (United States)

    Eitel, Alexander; Scheiter, Katharina

    2015-01-01

    The present article reviews 42 studies investigating the role of sequencing of text and pictures for learning outcomes. Whereas several of the reviewed studies revealed better learning outcomes from presenting the picture before the text rather than after it, other studies demonstrated the opposite effect. Against the backdrop of theories on…

  17. Learning German Formulaic Sequences: The Effect of Two Attention-Drawing Techniques

    Science.gov (United States)

    Peters, Elke

    2012-01-01

    This article reports a small-scale study that investigated the effect of (1) an instructional method, viz. directing learners' attention to formulaic sequences (FS) in a text, and (2) typographic salience, i.e. bold typeface and underlined, on foreign-language (FL) learners' recall of FS and single words (SW). Twenty-eight FL learners read a…

  18. Effects of Representation Sequences and Spatial Ability on Students' Scientific Understandings about the Mechanism of Breathing

    Science.gov (United States)

    Wu, Hsin-Kai; Lin, Yu-Fen; Hsu, Ying-Shao

    2013-01-01

    The purpose of this study was to investigate the effects of representation sequences and spatial ability on students' scientific understandings about the mechanism of breathing in human beings. 130 seventh graders were assigned to two groups with different sequential combinations of static and dynamic representations: SD group (i.e., viewing…

  19. The Effects of Method of Hierarchical Organization and Sequence on Children's Learning.

    Science.gov (United States)

    Parker, DeAnsin Goodson

    This study focused on Robert Gagne's method for curricular development, which consists of structuring a knowledge domain into a learning hierarchy. Two methods of generating learning hierarchies and two different sequencings of these hierarchies were compared and their effects were measured. Four programmed texts were developed from two different…

  20. Effects of Representation Sequences and Spatial Ability on Students' Scientific Understandings about the Mechanism of Breathing

    Science.gov (United States)

    Wu, Hsin-Kai; Lin, Yu-Fen; Hsu, Ying-Shao

    2013-01-01

    The purpose of this study was to investigate the effects of representation sequences and spatial ability on students' scientific understandings about the mechanism of breathing in human beings. 130 seventh graders were assigned to two groups with different sequential combinations of static and dynamic representations: SD group (i.e., viewing…

  1. Effective Simulation of Quantum Entanglement Based on Classical Fields Modulated with Pseudorandom Phase Sequences

    CERN Document Server

    Fu, Jian; Xu, Yingying; Dong, Hongtao

    2010-01-01

    We demonstrate that n classical fields modulated with n different pseudorandom phase sequences can constitute a 2^n-dimensional Hilbert space that contains tensor product structure. By using classical fields modulated with pseudorandom phase sequences, we discuss effective simulation of Bell states and GHZ state, and apply both correlation analysis and von Neumann entropy to characterize the simulation. We obtain similar results with the cases in quantum mechanics and find that the conclusions can be easily generalized to n quantum particles. The research on simulation of quantum entanglement may be important, for it not only provides useful insights into fundamental features of quantum entanglement, but also yields new insights into quantum computation.

  2. Does order matter? Investigating the effect of sequence on glance duration during on-road driving

    Science.gov (United States)

    Roberts, Shannon C.; Reimer, Bryan; Mehler, Bruce

    2017-01-01

    Previous literature has shown that vehicle crash risks increases as drivers’ off-road glance duration increases. Many factors influence drivers’ glance duration such as individual differences, driving environment, or task characteristics. Theories and past studies suggest that glance duration increases as the task progresses, but the exact relationship between glance sequence and glance durations is not fully understood. The purpose of this study was to examine the effect of glance sequence on glance duration among drivers completing a visual-manual radio tuning task and an auditory-vocal based multi-modal navigation entry task. Eighty participants drove a vehicle on urban highways while completing radio tuning and navigation entry tasks. Forty participants drove under an experimental protocol that required three button presses followed by rotation of a tuning knob to complete the radio tuning task while the other forty participants completed the task with one less button press. Multiple statistical analyses were conducted to measure the effect of glance sequence on glance duration. Results showed that across both tasks and a variety of statistical tests, glance sequence had inconsistent effects on glance duration—the effects varied according to the number of glances, task type, and data set that was being evaluated. Results suggest that other aspects of the task as well as interface design effect glance duration and should be considered in the context of examining driver attention or lack thereof. All in all, interface design and task characteristics have a more influential impact on glance duration than glance sequence, suggesting that classical design considerations impacting driver attention, such as the size and location of buttons, remain fundamental in designing in-vehicle interfaces. PMID:28158301

  3. Effect of k-tuple length on sample-comparison with high-throughput sequencing data.

    Science.gov (United States)

    Wang, Ying; Lei, Xiaoye; Wang, Shun; Wang, Zicheng; Song, Nianfeng; Zeng, Feng; Chen, Ting

    2016-01-22

    The high-throughput metagenomic sequencing offers a powerful technique to compare the microbial communities. Without requiring extra reference sequences, alignment-free models with short k-tuple (k = 2-10 bp) yielded promising results. Short k-tuples describe the overall statistical distribution, but is hard to capture the specific characteristics inside one microbial community. Longer k-tuple contains more abundant information. However, because the frequency vector of long k-tuple(k ≥ 30 bp) is sparse, the statistical measures designed for short k-tuples are not applicable. In our study, we considered each tuple as a meaningful word and then each sequencing data as a document composed of the words. Therefore, the comparison between two sequencing data is processed as "topic analysis of documents" in text mining. We designed a pipeline with long k-tuple features to compare metagenomic samples combined using algorithms from text mining and pattern recognition. The pipeline is available at http://culotuple.codeplex.com/. Experiments show that our pipeline with long k-tuple features: ①separates genomes with high similarity; ②outperforms short k-tuple models in all experiments. When k ≥ 12, the short k-tuple measures are not applicable anymore. When k is between 20 and 40, long k-tuple pipeline obtains much better grouping results; ③is free from the effect of sequencing platforms/protocols. ③We obtained meaningful and supported biological results on the 40-tuples selected for comparison.

  4. Primer effect in the detection of mitochondrial DNA point heteroplasmy by automated sequencing.

    Science.gov (United States)

    Calatayud, Marta; Ramos, Amanda; Santos, Cristina; Aluja, Maria Pilar

    2013-06-01

    The correct detection of mitochondrial DNA (mtDNA) heteroplasmy by automated sequencing presents methodological constraints. The main goals of this study are to investigate the effect of sense and distance of primers in heteroplasmy detection and to test if there are differences in the accurate determination of heteroplasmy involving transitions or transversions. A gradient of the heteroplasmy levels was generated for mtDNA positions 9477 (transition G/A) and 15,452 (transversion C/A). Amplification and subsequent sequencing with forward and reverse primers, situated at 550 and 150 bp from the heteroplasmic positions, were performed. Our data provide evidence that there is a significant difference between the use of forward and reverse primers. The forward primer is the primer that seems to give a better approximation to the real proportion of the variants. No significant differences were found concerning the distance at which the sequencing primers were placed neither between the analysis of transitions and transversions. The data collected in this study are a starting point that allows to glimpse the importance of the sequencing primers in the accurate detection of point heteroplasmy, providing additional insight into the overall automated sequencing strategy.

  5. DEVELOPMENT OF STEP-POOL SEQUENCE AND ITS EFFECTS IN RESISTANCE AND STREAM BED STABILITY

    Institute of Scientific and Technical Information of China (English)

    Zhao-Yin WANG; Jiang XU; Changzhi LI

    2004-01-01

    Experiments were conducted and field investigations were performed to study the development of step-pool sequence and its effects on resistance to the flow and stream bed stability. Step-pool sequence develops in incised channels as a result of streambed erosion, which is compared with sand dunes and armor layer of the role in resistance and streambed protection. The tight interlocking of particles in steps gives them an inherent stability which only extreme floods are likely to disturb. That stability suggests that step-pools are a valid equilibrium form, especially when coupled with their apparent regularity form and their role in satisfying the extreme condition of resistance maximization. The development degree of step-pools, SP, is proportional to the streambed slope. If the incoming sediment load is equal to or more than the sediment-carrying capacity of the flow, there is no bed erosion and thence there are no step-pools. Ifthe flow depth increases and is over the step-height the resistance caused by the step-pool sequence will be greatly reduced. The rate of energy dissipation by step-pools is a function of SP. The higher is SP, the larger is the rate of energy dissipation. The step-pool sequence increases the resistance and flow depth, reduces the shear stress of the flow and protects the streambed from erosion. Moreover,step-pool sequence provides ecologically sound habitats for aquatic bio-community as well.

  6. Effects of flanking base sequences on 5-bromodeoxyuridine mutagenesis in mammalian cells.

    Science.gov (United States)

    Kresnak, M T; Davidson, R L

    1991-07-01

    The molecular mechanisms of incorporation-dependent, 5-bromodeoxyuridine (BrdU)-induced mutagenesis were analyzed in murine A9 cells that possess a single copy of the Escherichia coli gpt gene integrated into the chromosomal DNA as part of a shuttle vector. Four independently derived GPT- mutants with single base changes within the integrated gpt gene were utilized in BrdU-induced reversion analyses to test the relative mutability of guanine residues in four different settings: the 5' and 3' guanine residues of a GG doublet, the 3' guanine residue of a GGGG quartet, and the middle guanine residue of a GGG triplet. Two of the mutant lines possessed GG doublet sequences in which a GC----AT transition at either guanine residue of the doublet leads to restoration of GPT enzyme activity without restoring wild-type DNA sequence. Both lines were shown to be effectively reverted by BrdU incorporation-dependent mutagenesis, and sequencing of the gpt genes from numerous independently derived revertants of both lines demonstrated that greater than 90% of the revertants arose due to GC----AT transitions at the 3' guanine residue of the doublet. BrdU-induced reversion of two additional GPT- mutant lines demonstrated that the 3' guanine residue of a GGGG quartet is efficiently mutated, while the middle guanine residue of a GGG triplet sequence is at least 10-fold less mutable by BrdU incorporation-dependent mutagenesis than the 3' guanine residue of a GG doublet or GGGG quartet. All four mutant lines tested were equally revertible by treatment with the alkylating agent ethyl methane sulfonate. The results from this study define a sequence-specific mechanism for BrdU-induced, incorporation-dependent mutagenesis and demonstrate the use of reversion analysis for the determination of sequence specific effects at precise sites within a gene.

  7. Development of a high-throughput G4-FID assay for screening and evaluation of small molecules binding quadruplex nucleic acid structures.

    Science.gov (United States)

    Largy, Eric; Hamon, Florian; Teulade-Fichou, Marie-Paule

    2011-07-01

    G4-FID (G-quadruplex fluorescent intercalator displacement) is a simple and fast method that allows to evaluate the affinity of a compound for G-quadruplex DNA and its selectivity towards duplex DNA. This assay is based on the loss of fluorescence of thiazole orange (TO) upon competitive displacement from DNA by a putative ligand. We describe here the development of a high-throughput version of this assay performed in 96-well microplates, and fully transposable to 384-well microplates. The test was calibrated with a set of G-quadruplex ligands characterized for their ability to bind quadruplex within a large range of affinity. The comparison of the results obtained in microplates and in cuvettes was conducted indicating a full agreement. Additionally, the spectral range of the test was enlarged using two other fluorescent on/off probes whose absorption are red-shifted (TO-PRO-3) and blue-shifted (Hoechst 33258) as compared to that of TO. These labels enable to screen a large diversity of compounds with various optical properties, which was exemplified by evaluation of affinity and selectivity of the porphyrin TMPyP4 that could not be evaluated previously. Altogether, our study demonstrates that the HT-G4-FID assay offers the possibility to label a large variety of G-quadruplexes of biological interest and should enable screening of collections of putative G4-ligands of high structural diversity. It thus represents a powerful tool to bring into light new ligands able to discriminate between quadruplexes of different structures.

  8. Modeling genetic imprinting effects of DNA sequences with multilocus polymorphism data

    Directory of Open Access Journals (Sweden)

    Staud Roland

    2009-08-01

    Full Text Available Abstract Single nucleotide polymorphisms (SNPs represent the most widespread type of DNA sequence variation in the human genome and they have recently emerged as valuable genetic markers for revealing the genetic architecture of complex traits in terms of nucleotide combination and sequence. Here, we extend an algorithmic model for the haplotype analysis of SNPs to estimate the effects of genetic imprinting expressed at the DNA sequence level. The model provides a general procedure for identifying the number and types of optimal DNA sequence variants that are expressed differently due to their parental origin. The model is used to analyze a genetic data set collected from a pain genetics project. We find that DNA haplotype GAC from three SNPs, OPRKG36T (with two alleles G and T, OPRKA843G (with alleles A and G, and OPRKC846T (with alleles C and T, at the kappa-opioid receptor, triggers a significant effect on pain sensitivity, but with expression significantly depending on the parent from which it is inherited (p = 0.008. With a tremendous advance in SNP identification and automated screening, the model founded on haplotype discovery and statistical inference may provide a useful tool for genetic analysis of any quantitative trait with complex inheritance.

  9. Glucose oxidase-initiated cascade catalysis for sensitive impedimetric aptasensor based on metal-organic frameworks functionalized with Pt nanoparticles and hemin/G-quadruplex as mimicking peroxidases.

    Science.gov (United States)

    Zhou, Xingxing; Guo, Shijing; Gao, Jiaxi; Zhao, Jianmin; Xue, Shuyan; Xu, Wenju

    2017-12-15

    Based on cascade catalysis amplification driven by glucose oxidase (GOx), a sensitive electrochemical impedimetric aptasensor for protein (carcinoembryonic antigen, CEA as tested model) was proposed by using Cu-based metal-organic frameworks functionalized with Pt nanoparticles, aptamer, hemin and GOx (Pt@CuMOFs-hGq-GOx). CEA aptamer loaded onto Pt@CuMOFs was bound with hemin to form hemin@G-quadruplex (hGq) with mimicking peroxidase activity. Through sandwich-type reaction of target CEA and CEA aptamers (Apt1 and Apt2), the obtained Pt@CuMOFs-hGq-GOx as signal transduction probes (STPs) was captured to the modified electrode interface. When 3,3-diaminobenzidine (DAB) and glucose were introduced, the cascade reaction was initiated by GOx to catalyze the oxidation of glucose, in situ generating H2O2. Simultaneously, the decomposition of the generated H2O2 was greatly promoted by Pt@CuMOFs and hGq as synergistic peroxide catalysts, accompanying with the significant oxidation process of DAB and the formation of nonconductive insoluble precipitates (IPs). As a result, the electron transfer in the resultant sensing interface was effectively hindered and the electrochemical impedimetric signal (EIS) was efficiently amplified. Thus, the high sensitivity of the proposed CEA aptasensor was successfully improved with 0.023pgmL(-1), which may be promising and potential in assaying certain clinical disease related to CEA. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Rapid three-dimensional microfluidic mixer for high viscosity solutions to unravel earlier folding kinetics of G-quadruplex under molecular crowding conditions.

    Science.gov (United States)

    Liu, Chao; Li, Ying; Li, Yiwei; Chen, Peng; Feng, Xiaojun; Du, Wei; Liu, Bi-Feng

    2016-01-01

    Rapid mixing of highly viscous solutions is a great challenge, which helps to analyze the reaction kinetics in viscous liquid phase, particularly to discover the folding kinetics of macromolecules under molecular crowding conditions mimicking the conditions inside cells. Here, we demonstrated a novel microfluidic mixer based on Dean flows with three-dimensional (3D) microchannel configuration for fast mixing of high-viscosity fluids. The main structure contained three consecutive subunits, each consisting of a "U"-type channel followed by a chamber with different width and height. Thus, the two solutions injected from the two inlets would undergo a mixing in the first "U"-type channel due to the Dean flow effect, and simultaneous vortices expansions in both horizontal and vertical directions in the following chamber. Numerical simulations and experimental characterizations confirmed that the micromixer could achieve a mixing time of 122.4μs for solutions with viscosities about 33.6 times that of pure water. It was the fastest micromixer for high viscosity solutions compared with previous reports. With this highly efficient 3D microfluidic mixer, we further characterized the early folding kinetics of human telomere G-quadruplex under molecular crowding conditions, and unravelled a new folding process within 550μs.

  11. Dispersed dynamics of solvation in G-quadruplex DNA: comparison of dynamic Stokes shifts of probes in parallel and antiparallel quadruplex structures

    Science.gov (United States)

    Kiran Singh, Moirangthem; Shweta, Him; Sen, Sobhan

    2016-09-01

    G-quadruplex DNA (GqDNA) structures play an important role in many specific cellular functions and are promising anti-tumor targets for small molecules (ligands). Here, we measured the dynamic Stokes shift of a ligand (Hoechst) bound to parallel c-Myc (mPu22) GqDNA over five decades of time from 100 fs to 10 ns, and compared it with the previously reported dynamics of DAPI bound to antiparallel human telomeric (hTelo22) GqDNA (Pal et al 2015 J. Phys. Chem. Lett. 6 1754). Stokes shift data from fluorescence up-conversion and time-correlated single photon counting experiments was combined to cover the broad dynamic range. The results show that the solvation dynamics of Hoechst in parallel mPu22 GqDNA follow a power law relaxation, added to fast 2 ps exponential relaxation, from 100 fs to 10 ns, with only a subtle difference of power law exponents in the two ligand-GqDNA systems (0.06 in Hoechst-mPu22 compared to 0.16 in DAPI-hTelo22). We measured steady-state fluorescence spectra and time-resolved anisotropy decays which confirm the tight binding of Hoechst to parallel mPu22 with a binding constant of ~1  ×  105 M-1. The molecular docking of Hoechst in parallel GqDNA followed by a 50 ns molecular dynamics (MD) simulation on a Hoechst-GqDNA complex reveals that Hoechst binds to one of the outer G-tetrads by end-stacking near G13 and G4, which is different from the binding site of DAPI inside a groove of antiparallel hTelo22 GqDNA. Reconciling previous experimental and simulation results, we assign the 2 ps component to the hydration dynamics of only weakly perturbed water near mPu22 and the power law relaxation to the coupled motion of water and DNA (i.e. DNA backbone, unpaired bases and loops connecting G-tetrads) which come near the Hoechst inside parallel GqDNA.

  12. Principles and procedures of considering item sequence effects in the development of calibrated item pools: Conceptual analysis and empirical illustration

    Directory of Open Access Journals (Sweden)

    Safir Yousfi

    2012-12-01

    Full Text Available Item responses can be context-sensitive. Consequently, composing test forms flexibly from a calibrated item pool requires considering potential context effects. This paper focuses on context effects that are related to the item sequence. It is argued that sequence effects are not necessarily a violation of item response theory but that item response theory offers a powerful tool to analyze them. If sequence effects are substantial, test forms cannot be composed flexibly on the basis of a calibrated item pool, which precludes applications like computerized adaptive testing. In contrast, minor sequence effects do not thwart applications of calibrated item pools. Strategies to minimize the detrimental impact of sequence effects on item parameters are discussed and integrated into a nomenclature that addresses the major features of item calibration designs. An example of an item calibration design demonstrates how this nomenclature can guide the process of developing a calibrated item pool.

  13. Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures

    Science.gov (United States)

    Chatterjee, Sujoy; Zagelbaum, Jennifer; Savitsky, Pavel; Sturzenegger, Andreas; Huttner, Diana; Janscak, Pavel; Hickson, Ian D.; Gileadi, Opher; Rothenberg, Eli

    2014-11-01

    Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.

  14. Reprogramming the mechanism of action of chlorambucil by coupling to a G-quadruplex ligand.

    Science.gov (United States)

    Di Antonio, Marco; McLuckie, Keith I E; Balasubramanian, Shankar

    2014-04-23

    The nitrogen mustard Chlorambucil (Chl) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts, interstrand cross-links (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Conversely, intrastrand cross-links generated by Chl are efficiently repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel cross-linking agent that combines Chl with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low μM concentrations, without reactivity toward double- or single-stranded DNA. Since intramolecular G4s arise from a single DNA strand, we reasoned that preferential alkylation of such structures might prevent the generation of ICLs, while favoring intrastrand cross-links. We observed that PDS-Chl selectively impairs growth in cells genetically deficient in NER, but did not show any sensitivity to the repair gene BRCA2, involved in double-stranded break repair. Our findings suggest that G4 targeting of this clinically important alkylating agent alters the overall mechanism of action. These insights may inspire new opportunities for intervention in diseases specifically characterized by genetic impairment of NER, such as skin and testicular cancers.

  15. G-quadruplexes in pathogens: a common route to virulence control?

    Science.gov (United States)

    Harris, Lynne M; Merrick, Catherine J

    2015-02-01

    DNA can form several secondary structures besides the classic double helix: one that has received much attention in recent years is the G-quadruplex (G4). This is a stable four-stranded structure formed by the stacking of quartets of guanine bases. Recent work has convincingly shown that G4s can form in vivo as well as in vitro and can affect both replication and transcription of DNA. They also play important roles at G-rich telomeres. Now, a spate of exciting reports has begun to reveal roles for G4 structures in virulence processes in several important microbial pathogens of humans. Interestingly, these come from a range of kingdoms--bacteria and protozoa as well as viruses--and all facilitate immune evasion in different ways. In particular, roles for G4s have been posited in the antigenic variation systems of bacteria and protozoa, as well as in the silencing of at least two major human viruses, human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV). Although antigenic variation and the silencing of latent viruses are quite distinct from one another, both are routes to immune evasion and the maintenance of chronic infections. Thus, highly disparate pathogens can use G4 motifs to control DNA/RNA dynamics in ways that are relevant to common virulence phenotypes. This review explores the evidence for G4 biology in such processes across a range of important human pathogens.

  16. Conformational conversion of DNA G-quadruplex induced by a cationic porphyrin.

    Science.gov (United States)

    Zhang, Huijuan; Xiao, Xiao; Wang, Peng; Pang, Siping; Qu, Feng; Ai, Xicheng; Zhang, Jianping

    2009-09-15

    The interactions between cationic meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the G-quadruplex (G4) of human telomeric single-strand oligonucleotide d(TTAGGG)(2) (S12) have been investigated by means of circular dichroism (CD), UV-visible absorption and fluorescence spectroscopies. It is found that TMPyP4 can preferentially induce the conformational conversion of the G4 structure from the parallel type to the parallel/antiparallel mixture in the presence of K(+), and that it can directly induce the formation of antiparallel G4 structure from the single-strand oligonucleotide S12 in the absence of K(+). Furthermore, the comparable experiments of TMPyP4 with two single-strand oligonucleotides S6 d(TTAGGG) and S24 d(TAGGG(TTAGGG)(3)T) in the absence of K(+) show that TMPyP4 can also induce the formation of antiparallel G4 from S24 but not from S6, indicating that the end-loops of the G4 structure are the key factors for the formation of G4 induced by TMPyP4.

  17. Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug screening.

    Science.gov (United States)

    Shen, Haihui; Zhang, Bo; Xu, Huiyan; Sun, Yue; Wu, Qiwang; Shen, Hong; Liu, Yingchun

    2017-02-05

    Some natural heterocyclic alkaloids containing planar group show potential to complex with specific promoter region of protooncogene for stabilizing the G-quadruplex (G4) structure which nowadays promises to be a target in anticancer drug design. However, in view of the polymorphic characteristics and structural complexity of heterocyclic alkaloids, it is desirable to develop high-throughput and low-consumption approach for anticancer drug screening. In this paper, an intensive study on alkaloid ligand/G4 DNA interaction has been conducted, demonstrating that the end-stacking interaction is the favorable binding mode between the oncogene-related Pu22 G4 DNA and the heterocyclic alkaloid ligand. Based on structural feasibility and energy minimization, a ligand displacement assay for screening alkaloid ligand in stabilizing the oncogene target G4 has been developed, which also helps to facilitate the assessment of drug specificity. Coupled with microfluidic-based DNAzyme-catalytic chemiluminescence detection, the approach showed the advantages of high sensitivity, high throughput with low sample and reagent consumptions.

  18. G-quadruplexes in pathogens: a common route to virulence control?

    Directory of Open Access Journals (Sweden)

    Lynne M Harris

    2015-02-01

    Full Text Available DNA can form several secondary structures besides the classic double helix: one that has received much attention in recent years is the G-quadruplex (G4. This is a stable four-stranded structure formed by the stacking of quartets of guanine bases. Recent work has convincingly shown that G4s can form in vivo as well as in vitro and can affect both replication and transcription of DNA. They also play important roles at G-rich telomeres. Now, a spate of exciting reports has begun to reveal roles for G4 structures in virulence processes in several important microbial pathogens of humans. Interestingly, these come from a range of kingdoms--bacteria and protozoa as well as viruses--and all facilitate immune evasion in different ways. In particular, roles for G4s have been posited in the antigenic variation systems of bacteria and protozoa, as well as in the silencing of at least two major human viruses, human immunodeficiency virus (HIV and Epstein-Barr virus (EBV. Although antigenic variation and the silencing of latent viruses are quite distinct from one another, both are routes to immune evasion and the maintenance of chronic infections. Thus, highly disparate pathogens can use G4 motifs to control DNA/RNA dynamics in ways that are relevant to common virulence phenotypes. This review explores the evidence for G4 biology in such processes across a range of important human pathogens.

  19. A single molecule study of a fluorescently labeled telomestatin derivative and G-quadruplex interactions

    Science.gov (United States)

    Maleki, Parastoo; Ma, Yue; Iida, Keisuke; Nagasawa, Kazuo; Balci, Hamza

    2017-01-01

    The potential use of G-quadruplex (GQ) stabilizing small molecules as anti-cancer drugs has created a flurry of activity on various aspects of these molecules. Telomestatin and oxazole telomestatin derivatives (OTD) are some of the most prominent of such molecules, yet the underlying dynamics of their interactions with GQ and the extent of heterogeneities in these interactions are not known. We performed single molecule measurements to study binding kinetics, rotational freedom, and dwell time distributions of a Cy5-labeled OTD (L1Cy5–7OTD) as it interacted with several different GQ structures. Our measurements show that L1Cy5–7OTD dwells on more stable GQ for longer times and binds to such GQ with higher frequency. The dwell times showed a broad distribution, but were longer than a minute for a significant fraction of molecules (characteristic dwell time τ = 192 ± 15 s and τ = 98 ± 15 s for the more and less stable GQ, respectively). In addition, L1Cy5–7OTD might be able to bind to GQ in at least two different primary orientations and occasionally transition between these orientations. The dwell time in one of these orientations was significantly longer than that in the other one, suggesting different stabilities for different binding orientations. PMID:27899628

  20. The Human Telomere Sequence, (TTAGGG4, in the Absence and Presence of Cosolutes: A Spectroscopic Investigation

    Directory of Open Access Journals (Sweden)

    Vishal R. Sharma

    2014-01-01

    Full Text Available Historically, biophysical studies of nucleic acids have been carried out under near ideal conditions, i.e., low buffer concentration (e.g., 10 mM phosphate, pH 7, low ionic strength (e.g., 100 mM and, for optical studies, low concentrations of DNA (e.g., 1 × 10−6 M. Although valuable structural and thermodynamic data have come out of these studies, the conditions, for the most, part, are inadequate to simulate realistic cellular conditions. The increasing interest in studying biomolecules under more cellular-like conditions prompted us to investigate the effect of osmotic stress on the structural and thermodynamic properties of DNA oligomers containing the human telomere sequence (TTAGGG. Here, we report the characterization of (TTAGGG4 in potassium phosphate buffer with increasing percent PEG (polyethylene glycol or acetonitrile. In general, the presence of these cosolutes induces a conformational change from a unimolecular hybrid structure to a multimolecular parallel stranded structure. Hence, the structural change is accompanied with a change in the molecularity of quadruplex formation.

  1. Dissecting the roles of local packing density and longer-range effects in protein sequence evolution

    CERN Document Server

    Shahmoradi, Amir

    2015-01-01

    What are the structural determinants of protein sequence evolution? A number of site-specific structural characteristics have been proposed, most of which are broadly related to either the density of contacts or the solvent accessibility of individual residues. Most importantly, there has been disagreement in the literature over the relative importance of solvent accessibility and local packing density for explaining site-specific sequence variability in proteins. We show here that this discussion has been confounded by the definition of local packing density. The most commonly used measures of local packing, such as the contact number and the weighted contact number, represent by definition the combined effects of local packing density and longer-range effects. As an alternative, we here propose a truly local measure of packing density around a single residue, based on the Voronoi cell volume. We show that the Voronoi cell volume, when calculated relative to the geometric center of amino-acid side chains, be...

  2. High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation.

    Science.gov (United States)

    Kerkour, Abdelaziz; Marquevielle, Julien; Ivashchenko, Stefaniia; Yatsunyk, Liliya A; Mergny, Jean-Louis; Salgado, Gilmar F

    2017-05-12

    Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Flower bud transcriptome analysis of Sapium sebiferum (Linn. Roxb. and primary investigation of drought induced flowering: pathway construction and G-quadruplex prediction based on transcriptome.

    Directory of Open Access Journals (Sweden)

    Minglei Yang

    Full Text Available Sapium sebiferum (Linn. Roxb. (Chinese Tallow Tree is a perennial woody tree and its seeds are rich in oil which hold great potential for biodiesel production. Despite a traditional woody oil plant, our understanding on S. sebiferum genetics and molecular biology remains scant. In this study, the first comprehensive transcriptome of S. sebiferum flower has been generated by sequencing and de novo assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in S. sebiferum flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis, 161 unigenes have been identified in fatty acid (FA and triacylglycerol (TAG biosynthesis. Besides, the G-Quadruplexes in RNA of S. sebiferum also have been predicted. An interesting finding is that the stress-induced flowering was observed in S. sebiferum for the first time. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, GA1, AP2 and CRY2, accorded with stress-related genes, such as GRX50435 and PRXⅡ39562. This transcriptome provides functional genomic information for further research of S. sebiferum, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offers a useful database for the research of other Euphorbiaceae family plants.

  4. Effects of cloning and root-tip size on observations of fungal ITS sequences from Picea glauca roots.

    Science.gov (United States)

    Lindner, Daniel L; Banik, Mark T

    2009-01-01

    To better understand the effects of cloning on observations of fungal ITS sequences from Picea glauca (white spruce) roots two techniques were compared: (i) direct sequencing of fungal ITS regions from individual root tips without cloning and (ii) cloning and sequencing of fungal ITS regions from individual root tips. Effect of root tip size was investigated by selecting 20 small root tips (SRT, 1.0-2.0 mm long) and 20 large root tips (LRT, 5.0-6.0 mm long). DNA was isolated from each tip and PCR-amplified with fungal-specific primers. PCR reactions were divided into two portions, one of which was sequenced directly and one of which was cloned first followed by sequencing of 12 random clones. With direct sequencing all 20 SRT produced an identifiable sequence, while only 13 of 20 LRT (65%) yielded an identifiable sequence. With cloning and sequencing all 40 tips produced identifiable fungal ITS sequences regardless of size. Failure of direct sequencing in LRT was associated with the presence of multispecies assemblages. Cloning identified 18 taxa overall while direct sequencing identified four. Cloning was not affected by tip size and identified more taxa relative to direct sequencing, although cost and probability of observing lab-based contaminants (e.g., airborne or reagent-based) were higher. We suggest that standardized controls be run whenever clones are sequenced from environmental samples, including positive controls derived from pure cultures and negative controls that cover the entire extraction, amplification and cloning process. Additional studies on larger root segments and bulked samples are needed to determine whether cloning can detect fungi accurately and cost-effectively in complex environmental samples.

  5. The effect of cognitive aging on implicit sequence learning and dual tasking

    Directory of Open Access Journals (Sweden)

    Jochen eVandenbossche

    2014-02-01

    Full Text Available We investigated the influence of attentional demands on sequence-specific learning by means of the serial reaction time (SRT task (Nissen & Bullemer, 1987 in young (age 18-25 and aged (age 55-75 adults. Participants had to respond as fast as possible to a stimulus presented in one of four horizontal locations by pressing a key corresponding to the spatial position of the stimulus. During the training phase sequential blocks were accompanied by (1 no secondary task (single, (2 a secondary tone counting task (dual tone, or (3 a secondary shape counting task (dual shape. Both secondary tasks were administered to investigate whether low and high interference tasks interact with implicit learning and age. The testing phase, under baseline single condition, was implemented to assess differences in sequence-specific learning between young and aged adults. Results indicate that (1 aged subjects show less sequence learning compared to young adults, (2 young participants show similar implicit learning effects under both single and dual task conditions when we account for explicit awareness, and (3 aged adults demonstrate reduced learning when the primary task is accompanied with a secondary task, even when explicit awareness is included as a covariate in the analysis. These findings point to implicit learning deficits under dual task conditions that can be related to cognitive aging, demonstrating the need for sufficient cognitive resources while performing a sequence learning task.

  6. Effects of the antimicrobial tylosin on the microbial community structure of an anaerobic sequencing batch reactor.

    Science.gov (United States)

    Shimada, Toshio; Li, Xu; Zilles, Julie L; Morgenroth, Eberhard; Raskin, Lutgarde

    2011-02-01

    The effects of the antimicrobial tylosin on a methanogenic microbial community were studied in a glucose-fed laboratory-scale anaerobic sequencing batch reactor (ASBR) exposed to stepwise increases of tylosin (0, 1.67, and 167 mg/L). The microbial community structure was determined using quantitative fluorescence in situ hybridization (FISH) and phylogenetic analyses of bacterial 16S ribosomal RNA (rRNA) gene clone libraries of biomass samples. During the periods without tylosin addition and with an influent tylosin concentration of 1.67 mg/L, 16S rRNA gene sequences related to Syntrophobacter were detected and the relative abundance of Methanosaeta species was high. During the highest tylosin dose of 167 mg/L, 16S rRNA gene sequences related to Syntrophobacter species were not detected and the relative abundance of Methanosaeta decreased considerably. Throughout the experimental period, Propionibacteriaceae and high GC Gram-positive bacteria were present, based on 16S rRNA gene sequences and FISH analyses, respectively. The accumulation of propionate and subsequent reactor failure after long-term exposure to tylosin are attributed to the direct inhibition of propionate-oxidizing syntrophic bacteria closely related to Syntrophobacter and the indirect inhibition of Methanosaeta by high propionate concentrations and low pH. © 2010 Wiley Periodicals, Inc.

  7. Sequencing Effects of Balance and Plyometric Training on Physical Performance in Youth Soccer Athletes.

    Science.gov (United States)

    Hammami, Raouf; Granacher, Urs; Makhlouf, Issam; Behm, David G; Chaouachi, Anis

    2016-12-01

    Hammami, R, Granacher, U, Makhlouf, I, Behm, DG, and Chaouachi, A. Sequencing effects of balance and plyometric training on physical performance in youth soccer athletes. J Strength Cond Res 30(12): 3278-3289, 2016-Balance training may have a preconditioning effect on subsequent power training with youth. There are no studies examining whether the sequencing of balance and plyometric training has additional training benefits. The objective was to examine the effect of sequencing balance and plyometric training on the performance of 12- to 13-year-old athletes. Twenty-four young elite soccer players trained twice per week for 8 weeks either with an initial 4 weeks of balance training followed by 4 weeks of plyometric training (BPT) or 4 weeks of plyometric training proceeded by 4 weeks of balance training (PBT). Testing was conducted pre- and posttraining and included medicine ball throw; horizontal and vertical jumps; reactive strength; leg stiffness; agility; 10-, 20-, and 30-m sprints; Standing Stork balance test; and Y-Balance test. Results indicated that BPT provided significantly greater improvements with reactive strength index, absolute and relative leg stiffness, triple hop test, and a trend for the Y-Balance test (p = 0.054) compared with PBT. Although all other measures had similar changes for both groups, the average relative improvement for the BPT was 22.4% (d = 1.5) vs. 15.0% (d = 1.1) for the PBT. BPT effect sizes were greater with 8 of 13 measures. In conclusion, although either sequence of BPT or PBT improved jumping, hopping, sprint acceleration, and Standing Stork and Y-Balance, BPT initiated greater training improvements in reactive strength index, absolute and relative leg stiffness, triple hop test, and the Y-Balance test. BPT may provide either similar or superior performance enhancements compared with PBT.

  8. Distribution of zero sequence currents for earth faults occurring along a transmission line and proximity effects

    Energy Technology Data Exchange (ETDEWEB)

    Nahman, J. (Belgrade Univ. (Yugoslavia). Elektrotehnicki Fakultet); Dordevic, V. (Energoprojekt, Belgrade (Yugoslavia))

    1993-09-01

    A relatively simple procedure is suggested for the evaluation of the distribution of zero sequence currents, within the earthing system of a substation, for earth faults occurred along a line coming from the substation. The earthing system model derived takes into account all relevant phenomena including the mutual influence among earth electrodes through the soil to cover the proximity effects which were shown to be significant in certain cases. The procedure suggested is applied to a practical case, for illustration. (author)

  9. Effect of photoperiod on the feline adipose transcriptome as assessed by RNA sequencing

    OpenAIRE

    Mori, Akihiro; Kappen, Kelly L; Dilger, Anna C; Swanson, Kelly S.

    2014-01-01

    Background Photoperiod is known to cause physiological changes in seasonal mammals, including changes in body weight, physical activity, reproductive status, and adipose tissue gene expression in several species. The objective of this study was to determine the effects of day length on the adipose transcriptome of cats as assessed by RNA sequencing. Ten healthy adult neutered male domestic shorthair cats were used in a randomized crossover design study. During two 12-wk periods, cats were exp...

  10. Effect of sequence features on assembly of spider silk block copolymers.

    Science.gov (United States)

    Tokareva, Olena S; Lin, Shangchao; Jacobsen, Matthew M; Huang, Wenwen; Rizzo, Daniel; Li, David; Simon, Marc; Staii, Cristian; Cebe, Peggy; Wong, Joyce Y; Buehler, Markus J; Kaplan, David L

    2014-06-01

    Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. The Effect of Stress and Speech Rate on Vowel Coarticulation in Catalan Vowel-Consonant-Vowel Sequences

    Science.gov (United States)

    Recasens, Daniel

    2015-01-01

    Purpose: The goal of this study was to ascertain the effect of changes in stress and speech rate on vowel coarticulation in vowel-consonant-vowel sequences. Method: Data on second formant coarticulatory effects as a function of changing /i/ versus /a/ were collected for five Catalan speakers' productions of vowel-consonant-vowel sequences with the…

  12. Effect of Sequencing Strength and Endurance Training in Young Male Soccer Players.

    Science.gov (United States)

    Makhlouf, Issam; Castagna, Carlo; Manzi, Vincenzo; Laurencelle, Louis; Behm, David G; Chaouachi, Anis

    2016-03-01

    This study examined the effects of strength and endurance training sequence (strength before or after endurance) on relevant fitness variables in youth soccer players. Fifty-seven young elite-level male field soccer players (13.7 ± 0.5 years; 164 ± 8.3 cm; 53.5 ± 8.6 kg; body fat; 15.6 ± 3.9%) were randomly assigned to a control (n = 14, CG) and 3 experimental training groups (twice a week for 12 weeks) strength before (SE, n = 15), after (ES, n = 14) or on alternate days (ASE, n = 14) with endurance training. A significant (p = 0.001) intervention main effect was detected. There were only trivial training sequence differences (ES vs. SE) for all variables (p > 0.05). The CG showed large squat 1 repetition maximum (1RM) and medium sprint, change of direction ability, and jump improvements. ASE demonstrated a trivial difference in endurance performance with ES and SE (p > 0.05). Large to medium greater improvements for SE and ES were reported compared with ASE for sprinting over 10 and 30 m (p training sequence on soccer fitness-relevant variables. However, combining strength and endurance within a single training session provided superior results vs. training on alternate days. Concurrent training may be considered as an effective and safe training method for the development of the prospective soccer player.

  13. Speech serial control in healthy speakers and speakers with hypokinetic or ataxic dysarthria: effects of sequence length and practice.

    Science.gov (United States)

    Reilly, Kevin J; Spencer, Kristie A

    2013-01-01

    The current study investigated the processes responsible for selection of sounds and syllables during production of speech sequences in 10 adults with hypokinetic dysarthria from Parkinson's disease, five adults with ataxic dysarthria, and 14 healthy control speakers. Speech production data from a choice reaction time task were analyzed to evaluate the effects of sequence length and practice on speech sound sequencing. Speakers produced sequences that were between one and five syllables in length over five experimental runs of 60 trials each. In contrast to the healthy speakers, speakers with hypokinetic dysarthria demonstrated exaggerated sequence length effects for both inter-syllable intervals (ISIs) and speech error rates. Conversely, speakers with ataxic dysarthria failed to demonstrate a sequence length effect on ISIs and were also the only group that did not exhibit practice-related changes in ISIs and speech error rates over the five experimental runs. The exaggerated sequence length effects in the hypokinetic speakers with Parkinson's disease are consistent with an impairment of action selection during speech sequence production. The absent length effects observed in the speakers with ataxic dysarthria is consistent with previous findings that indicate a limited capacity to buffer speech sequences in advance of their execution. In addition, the lack of practice effects in these speakers suggests that learning-related improvements in the production rate and accuracy of speech sequences involves processing by structures of the cerebellum. Together, the current findings inform models of serial control for speech in healthy speakers and support the notion that sequencing deficits contribute to speech symptoms in speakers with hypokinetic or ataxic dysarthria. In addition, these findings indicate that speech sequencing is differentially impaired in hypokinetic and ataxic dysarthria.

  14. Speech serial control in healthy speakers and speakers with hypokinetic or ataxic dysarthria: Effects of sequence length and practice

    Directory of Open Access Journals (Sweden)

    Kevin J Reilly

    2013-10-01

    Full Text Available The current study investigated the processes responsible for selection of sounds and syllables during production of speech sequences in 10 adults with hypokinetic dysarthria from Parkinson’s disease, 5 adults with ataxic dysarthria, and 14 healthy control speakers. Speech production data from a choice reaction time task were analyzed to evaluate the effects of sequence length and practice on speech sound sequencing. Speakers produced sequences that were between one and five syllables in length over five experimental runs of 60 trials each. In contrast to the healthy speakers, speakers with hypokinetic dysarthria demonstrated exaggerated sequence length effects for both inter-syllable intervals (ISIs and speech error rates. Conversely, speakers with ataxic dysarthria failed to demonstrate a sequence length effect on ISIs and were also the only group that did not exhibit practice-related changes in ISIs and speech error rates over the five experimental runs. The exaggerated sequence length effects in the hypokinetic speakers with Parkinson’s disease are consistent with an impairment of action selection during speech sequence production. The absent length effects observed in the speakers with ataxic dysarthria is consistent with previous findings that indicate a limited capacity to buffer speech sequences in advance of their execution. In addition, the lack of practice effects in these speakers suggests that learning-related improvements in the production rate and accuracy of speech sequences involves processing by structures of the cerebellum. Together, the current findings inform models of serial control for speech in healthy speakers and support the notion that sequencing deficits contribute to speech symptoms in speakers with hypokinetic or ataxic dysarthria. In addition, these findings indicate that speech sequencing is differentially impaired in hypokinetic and ataxic dysarthria.

  15. The effect of music background on the emotional appraisal of film sequences

    Directory of Open Access Journals (Sweden)

    Pavlović Ivanka

    2011-01-01

    Full Text Available In this study the effects of musical background on the emotional appraisal of film sequences was investigated. Four pairs of polar emotions defined in Plutchik’s model were used as basic emotional qualities: joy-sadness, anticipation-surprise, fear-anger, and trust disgust. In the preliminary study eight film sequences and eight music themes were selected as the best representatives of all eight Plutchik’s emotions. In the main experiment the participant judged the emotional qualities of film-music combinations on eight seven-point scales. Half of the combinations were congruent (e.g. joyful film - joyful music, and half were incongruent (e.g. joyful film - sad music. Results have shown that visual information (film had greater effects on the emotion appraisal than auditory information (music. The modulation effects of music background depend on emotional qualities. In some incongruent combinations (joysadness the modulations in the expected directions were obtained (e.g. joyful music reduces the sadness of a sad film, in some cases (anger-fear no modulation effects were obtained, and in some cases (trust-disgust, anticipation-surprise the modulation effects were in an unexpected direction (e.g. trustful music increased the appraisal of disgust of a disgusting film. These results suggest that the appraisals of conjoint effects of emotions depend on the medium (film masks the music and emotional quality (three types of modulation effects.

  16. Extreme weather-year sequences have non-additive effects on environmental nitrogen losses.

    Science.gov (United States)

    Iqbal, Javed; Necpalova, Magdalena; Archontoulis, Sotirios V; Anex, Robert P; Bourguignon, Marie; Herzmann, Daryl; Mitchell, David C; Sawyer, John E; Zhu, Qing; Castellano, Michael J

    2017-08-14

    The frequency and intensity of extreme weather years, characterized by abnormal precipitation and temperature, are increasing. In isolation, these years have disproportionately large effects on environmental N losses. However, the sequence of extreme weather years (e.g., wet-dry vs. dry-wet) may affect cumulative N losses. We calibrated and validated the DAYCENT ecosystem process model with a comprehensive set of biogeophysical measurements from a corn-soybean rotation managed at three N fertilizer inputs with and without a winter cover crop in Iowa, USA. Our objectives were to determine: i) how two-year sequences of extreme weather affect two-year cumulative N losses across the crop rotation, and ii) if N fertilizer management and the inclusion of a winter cover crop between corn and soybean mitigate the effect of extreme weather on N losses. Using historical weather (1951-2013), we created nine two-year scenarios with all possible combinations of the driest ('dry'), wettest ('wet'), and average ('normal') weather years. We analyzed the effects of these scenarios following several consecutive years of relatively normal weather. Compared to the normal-normal two-year weather scenario, two-year extreme weather scenarios affected two-year cumulative NO3(-) leaching (range: -93 to +290%) more than N2 O emissions (range: -49 to +18%). The two-year weather scenarios had non-additive effects on N losses: compared to the normal-normal scenario, the dry-wet sequence decreased two-year cumulative N2 O emissions while the wet-dry sequence increased two-year cumulative N2 O emissions. Although dry weather decreased NO3(-) leaching and N2 O emissions in isolation, two-year cumulative N losses from the wet-dry scenario were greater than the dry-wet scenario. Cover crops reduced the effects of extreme weather on NO3(-) leaching but had a lesser effect on N2 O emissions. As the frequency of extreme weather is expected to increase, these data suggest that the sequence of inter

  17. Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies.

    Directory of Open Access Journals (Sweden)

    Patrick D Schloss

    Full Text Available The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10(6 reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially

  18. Fast and cost-effective genetic mapping in apple using next-generation sequencing.

    Science.gov (United States)

    Gardner, Kyle M; Brown, Patrick; Cooke, Thomas F; Cann, Scott; Costa, Fabrizio; Bustamante, Carlos; Velasco, Riccardo; Troggio, Michela; Myles, Sean

    2014-07-16

    Next-generation DNA sequencing (NGS) produces vast amounts of DNA sequence data, but it is not specifically designed to generate data suitable for genetic mapping. Recently developed DNA library preparation methods for NGS have helped solve this problem, however, by combining the use of reduced representation libraries with DNA sample barcoding to generate genome-wide genotype data from a common set of genetic markers across a large number of samples. Here we use such a method, called genotyping-by-sequencing (GBS), to produce a data set for genetic mapping in an F1 population of apples (Malus × domestica) segregating for skin color. We show that GBS produces a relatively large, but extremely sparse, genotype matrix: over 270,000 SNPs were discovered but most SNPs have too much missing data across samples to be useful for genetic mapping. After filtering for genotype quality and missing data, only 6% of the 85 million DNA sequence reads contributed to useful genotype calls. Despite this limitation, using existing software and a set of simple heuristics, we generated a final genotype matrix containing 3967 SNPs from 89 DNA samples from a single lane of Illumina HiSeq and used it to create a saturated genetic linkage map and to identify a known QTL underlying apple skin color. We therefore demonstrate that GBS is a cost-effective method for generating genome-wide SNP data suitable for genetic mapping in a highly diverse and heterozygous agricultural species. We anticipate future improvements to the GBS analysis pipeline presented here that will enhance the utility of next-generation DNA sequence data for the purposes of genetic mapping across diverse species.

  19. Exploring non-covalent interactions in guanine- and xanthine-based model DNA quadruplex structures: a comprehensive quantum chemical approach.

    Science.gov (United States)

    Yurenko, Yevgen P; Novotný, Jan; Sklenář, Vladimir; Marek, Radek

    2014-02-07

    The study aimed to cast light on the structure and internal energetics of guanine- and xanthine-based model DNA quadruplexes and the physico-chemical nature of the non-covalent interactions involved. Several independent approaches were used for this purpose: DFT-D3 calculations, Quantum Theory of Atoms in Molecules, Natural Bond Orbital Analysis, Energy Decomposition Analysis, Compliance Constant Theory, and Non-Covalent Interaction Analysis. The results point to an excellent degree of structural and energetic compatibility between the two types of model quadruplexes. This fact stems from both the structural features (close values of van der Waals volumes, pore radii, geometrical parameters of the H-bonds) and the energetic characteristics (comparable values of the energies of formation). It was established that hydrogen bonding makes the greatest (∼50%) contribution to the internal stability of the DNA quadruplexes, whereas the aromatic base stacking and ion coordination terms are commensurable and account for the rest. Energy decomposition analysis performed for guanine (Gua) and xanthine (Xan) quartets B4 and higher-order structures consisting of two or three stacked quartets indicates that whereas Gua structures benefit from a high degree of H-bond cooperativity, Xan models are characterized by a more favorable and cooperative π-π stacking. The results of electron density topological analysis show that Na(+)/K(+) ion coordination deeply affects the network of non-covalent interactions in Gua models due to the change in the twist angle between the stacked tetrads. For Xan models, ion coordination makes tetrads in stacks more planar without changing the twist angle. Therefore, the presence of the ion seems to be essential for the formation of planar stacks in Xan-based DNA quadruplexes. Detailed study of the nature of ion-base coordination suggests that this interaction has a partially covalent character and cannot be considered as purely electrostatic

  20. Molecular design of synthetic benzimidazoles for the switchover of the duplex to G-quadruplex DNA recognition.

    Science.gov (United States)

    Maji, Basudeb; Bhattacharya, Santanu

    2013-01-01

    Benzimidazole derivatives are well known for their antibacterial, antiviral, anticonvulsant, antihistaminic, anthelmintic and antidepressant activities. Benzimidazole's unique base-selective DNA recognition property has been studied widely. However, most of the early benzimidazole systems have been targeted towards the binding of duplex DNA. Here we have shown the evolution and progress of the design and synthesis of new benzimidazole systems towards selective recognition of the double-stranded DNA first. Then in order to achieve selective recognition of the G-quadruplex DNA and utilize their potential as future anti-cancer drug candidates, we have demonstrated their selective cytotoxicity towards the cancer cells and potent telomerase inhibition ability.

  1. The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell

    OpenAIRE

    Perrone, Rosalba; Butovskaya, Elena; Lago, Sara; Garzino-Demo, Alfredo; Pannecouque, Christophe; Palù, Giorgio; Richter, Sara N.

    2016-01-01

    AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HI...

  2. Quadruplex PCR for simultaneous detection of serotype, biotype, toxigenic potential, and central regulating factor of Vibrio cholerae.

    Science.gov (United States)

    Khuntia, Hemant Kumar; Pal, Bibhuti Bhusan; Chhotray, Guru Prasada

    2008-07-01

    A quadruplex PCR was developed for the simultaneous detection of genes specific for Vibrio cholerae O1 and/or O139 serogroup (wbe and/or wbf), cholera toxin A subunit (ctxA), toxin-coregulated pilus (tcpA), and central regulating protein ToxR (toxR) in a single tube reaction. This is a simple, rapid, and accurate approach for the detection of toxigenic V. cholerae O1 and/or O139 and can prevent the rapid spread of the disease by early detection.

  3. Novel computational methods for increasing PCR primer design effectiveness in directed sequencing

    Directory of Open Access Journals (Sweden)

    Busam Dana

    2008-04-01

    Full Text Available Abstract Background Polymerase chain reaction (PCR is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment. Results We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research. Conclusion The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing

  4. Effect of Impregnation Sequence on Propane Dehydrogenation Performance of PtSnNa/ZSM-5 Catalyst

    Institute of Scientific and Technical Information of China (English)

    Liu Hui; Zhou Yuming; Zhang Yiwei; Sheng Xiaoli; Zhang Zewu; Zhou Shijiang

    2013-01-01

    The effects of the sequence for impregnation of metal precursors on the performance of PtSnNa/ZSM-5 catalyst for propane dehydrogenation to propene were studied in this paper. Some methods such as XRD, TPDA,BET, H2-TPR, XPS, ICP, TEM and hydrogen chemisorption were used to characterize the catalysts. The structure of ZSM-5 zeolite was not destroyed by the introduction of metal components. Meanwhile the different impregnation sequence of metal precursors could affect the behavior of Sn4+ species entering the ZSM-5 channel, and the interaction between platinum and tin species, as well as the degree for reduction of Pt and Sn components. As a result, the prepared catalysts exhibited different reac-tion activity and selectivity. Compared with the co-impregnation treated catalyst, the catalysts prepared by the sequential impregnation method showed better catalytic activity in propane dehydrogenation, especially the one prepared through im-pregnation with tin precursor at ifrst. Finally, a model for the effect of impregnation sequence on the distribution of Pt and Sn species in PtSnNa/ZSM-5 catalyst was proposed.

  5. Effects of RNA sequence specificity in the electrostatic stabilization of viruses

    CERN Document Server

    Erdemci-Tandogan, Gonca; van der Schoot, Paul; Podgornik, Rudolf; Zandi, Roya

    2016-01-01

    Many single-stranded (ss) RNA viruses self assemble from capsid protein subunits and the nucleic acid to form an infectious virion. It is believed that the electrostatic interactions between the negatively charged RNA and the positively charged viral capsid proteins drive the encapsidation, although there is growing evidence that the sequence of the viral RNA also plays a role in packaging. In particular the sequence will determine the possible secondary structures that the ssRNA will take in solution. In this work, we use a mean field theory to investigate how the secondary structure of the RNA combined with electrostatic interactions affects the efficiency of assembly and stability of the assembled virions. We show that the secondary structure of RNA may result in negative osmotic pressures while a linear polymer causes positive osmotic pressures for the same conditions. This may suggest that the branched structure makes the RNA more effectively packaged and the virion more stable.

  6. Welding sequence effects on residual stress distribution in offshore wind monopile structures

    Directory of Open Access Journals (Sweden)

    Ali Mehmanparast

    2016-01-01

    Full Text Available Residual stresses are often inevitably introduced into the material during the fabrication processes, such as welding, and are known to have significant effects on the subsequent fatigue crack growth behavior of welded structures. In this paper, the importance of welding sequence on residual stress distribution in engineering components has been reviewed. In addition, the findings available in the literature have been used to provide an accurate interpretation of the fatigue crack growth data on specimens extracted from the welded plates employed in offshore wind monopile structures. The results have been discussed in terms of the role of welding sequence in damage inspection and structural integrity assessment of offshore renewable energy structures.

  7. STUDY OF BLOCKING EFFECT ELIMINATION METHODS BY MEANS OF INTRAFRAME VIDEO SEQUENCE INTERPOLATION

    Directory of Open Access Journals (Sweden)

    I. S. Rubina

    2015-01-01

    Full Text Available The paper deals with image interpolation methods and their applicability to eliminate some of the artifacts related to both the dynamic properties of objects in video sequences and algorithms used in the order of encoding steps. The main drawback of existing methods is the high computational complexity, unacceptable in video processing. Interpolation of signal samples for blocking - effect elimination at the output of the convertion encoding is proposed as a part of the study. It was necessary to develop methods for improvement of compression ratio and quality of the reconstructed video data by blocking effect elimination on the borders of the segments by intraframe interpolating of video sequence segments. The main point of developed methods is an adaptive recursive algorithm application with adaptive-sized interpolation kernel both with and without the brightness gradient consideration at the boundaries of objects and video sequence blocks. Within theoretical part of the research, methods of information theory (RD-theory and data redundancy elimination, methods of pattern recognition and digital signal processing, as well as methods of probability theory are used. Within experimental part of the research, software implementation of compression algorithms with subsequent comparison of the implemented algorithms with the existing ones was carried out. Proposed methods were compared with the simple averaging algorithm and the adaptive algorithm of central counting interpolation. The advantage of the algorithm based on the adaptive kernel size selection interpolation is in compression ratio increasing by 30%, and the advantage of the modified algorithm based on the adaptive interpolation kernel size selection is in the compression ratio increasing by 35% in comparison with existing algorithms, interpolation and quality of the reconstructed video sequence improving by 3% compared to the one compressed without interpolation. The findings will be

  8. Sequence context of indel mutations and their effect on protein evolution in a bacterial endosymbiont.

    Science.gov (United States)

    Williams, Laura E; Wernegreen, Jennifer J

    2013-01-01

    Indel mutations play key roles in genome and protein evolution, yet we lack a comprehensive understanding of how indels impact evolutionary processes. Genome-wide analyses enabled by next-generation sequencing can clarify the context and effect of indels, thereby integrating a more detailed consideration of indels with our knowledge of nucleotide substitutions. To this end, we sequenced Blochmannia chromaiodes, an obligate bacterial endosymbiont of carpenter ants, and compared it with the close relative, B. pennsylvanicus. The genetic distance between these species is small enough for accurate whole genome alignment but large enough to provide a meaningful spectrum of indel mutations. We found that indels are subjected to purifying selection in coding regions and even intergenic regions, which show a reduced rate of indel base pairs per kilobase compared with nonfunctional pseudogenes. Indels occur almost exclusively in repeat regions composed of homopolymers and multimeric simple sequence repeats, demonstrating the importance of sequence context for indel mutations. Despite purifying selection, some indels occur in protein-coding genes. Most are multiples of three, indicating selective pressure to maintain the reading frame. The deleterious effect of frameshift-inducing indels is minimized by either compensation from a nearby indel to restore reading frame or the indel's location near the 3'-end of the gene. We observed amino acid divergence exceeding nucleotide divergence in regions affected by frameshift-inducing indels, suggesting that these indels may either drive adaptive protein evolution or initiate gene degradation. Our results shed light on how indel mutations impact processes of molecular evolution underlying endosymbiont genome evolution.

  9. A label-free fluorescence DNA probe based on ligation reaction with quadruplex formation for highly sensitive and selective detection of nicotinamide adenine dinucleotide.

    Science.gov (United States)

    Zhao, Jingjin; Zhang, Liangliang; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

    2012-05-11

    A simple label-free fluorescent sensing scheme for sensitive and selective detection of nicotinamide adenine dinucleotide (NAD(+)) has been developed based on DNA ligation reaction with ligand-responsive quadruplex formation. This approach can detect 0.5 nM NAD(+) with high selectivity against other NAD(+) analogs.

  10. Amplified detection of DNA ligase and polynucleotide kinase/phosphatase on the basis of enrichment of catalytic G-quadruplex DNAzyme by rolling circle amplification.

    Science.gov (United States)

    Jiang, Hong-Xin; Kong, De-Ming; Shen, Han-Xi

    2014-05-15

    As two commonly used tool enzymes, DNA ligase and polynucleotide kinase/phosphatase (PNKP) play important roles in DNA metabolism. More and more studies show that regulation of their activity represents promising means for cancer therapy. To detect the activity of DNA ligase with high sensitivity and specificity, a G-quadruplex DNAzyme-based DNA ligase sensor was developed. In this sensor, the use of G-quadruplex DNAzyme eliminated the needs for any labeled oligonucleotide probes, thus making label-free detection possible. The introduction of rolling circle amplification (RCA) reaction could lead to the formation of multimeric G-quadruplexes containing thousands of G-quadruplex units, which can provide highly active hemin-binding sites, thus significantly improving the sensitivity of the sensor. The proposed sensor allowed specific detection of T4 DNA ligase with a detection limit of 0.0019 U/mL. By adding a PNKP-triggered 5'-phosphroylation step of the template DNA, the above sensing strategy could be easily extended to the design of PNKP sensor. The established sensor allowed specific detection of T4 PNKP with a detection limit of 0.0018 U/mL. In addition, these two sensors could also be used for the studies on inhibitors of these two enzymes.

  11. The high-resolution crystal structure of a parallel intermolecular DNA G-4 quadruplex/drug complex employing syn glycosyl linkages

    Science.gov (United States)

    Clark, George R.; Pytel, Patrycja D.; Squire, Christopher J.

    2012-01-01

    We have determined the X-ray structure of the complex between the DNA quadruplex d(5′-GGGG-3′)4 and daunomycin, as a potential model for studying drug–telomere interactions. The structure was solved at 1.08 Å by direct methods in space group I4. The asymmetric unit comprises a linear arrangement of one d(GGGG) strand, four daunomycin molecules, a second d(GGGG) strand facing in the opposite direction to the first, and Na and Mg cations. The crystallographic 4-fold axis generates the biological unit, which is a 12-layered structure comprising two sets of four guanine layers, with four layers each of four daunomycins stacked between the 5′ faces of the two quadruplexes. The daunomycin layers fall into two groups which are novel in their mode of self assembly. The only contacts between daunomycin molecules within any one of these layers are van der Waals interactions, however there is substantial π–π stacking between successive daunomycin layers and also with adjacent guanine layers. The structure differs significantly from all other parallel d(TGGGGT)4 quadruplexes in that the 5′ guanine adopts the unusual syn glycosyl linkage, refuting the widespread belief that such conformations should all be anti. In contrast to the related d(TGGGGT)/daunomycin complex, there are no ligand–quadruplex groove insertion interactions. PMID:22373921

  12. A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires.

    Science.gov (United States)

    Xie, Shunbi; Chai, Yaqin; Yuan, Yali; Bai, Lijuan; Yuan, Ruo

    2014-06-17

    In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR's ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H2O2. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1 pM-50 nM with a detection limit of 33 fM (defined as S/N=3) for TB.

  13. Porous platinum nanotubes labeled with hemin/G-quadruplex based electrochemical aptasensor for sensitive thrombin analysis via the cascade signal amplification.

    Science.gov (United States)

    Sun, Aili; Qi, Qingan; Wang, Xuannian; Bie, Ping

    2014-07-15

    For the first time, a sensitive electrochemical aptasensor for thrombin (TB) was developed by using porous platinum nanotubes (PtNTs) labeled with hemin/G-quadruplex and glucose dehydrogenase (GDH) as labels. Porous PtNTs with large surface area exhibited the peroxidase-like activity. Coupling with GDH and hemin/G-quadruplex as NADH oxidase and HRP-mimicking DNAzyme, the cascade signal amplification was achieved by the following ways: in the presence of glucose and NAD(+) in the working buffer, GDH electrocatalyzed the oxidation of glucose with the production of NADH. Then, hemin/G-quadruplex as NADH oxidase catalyzed the oxidation of NADH to in situ generate H2O2. Based on the corporate electrocatalysis of PtNTs and hemin/G-quadruplex toward H2O2, the electrochemical signal was significantly amplified, allowing the detection limit of TB down to 0.15 pM level. Moreover, the proposed strategy was simple because the intercalated hemin offered the readout signal, avoiding the adding of additional redox mediator as signal donator. Such an electrochemical aptasensor is highly promising for sensitive detection of other proteins in clinical diagnostics.

  14. Voltammetry and Molecular Assembly of G-quadruplex DNAzyme on Single-crystal Au(111)-electrode Surfaces – Hemin as an Electrochemical Intercalator

    DEFF Research Database (Denmark)

    Zhang, Ling; Ulstrup, Jens; Zhang, Jingdong

    2016-01-01

    DNA quadruplexes (qs’s) are a class of “non-canonical” oligonucleotides (OGNs) composed of stacked guanine (G) quartets generally stabilized by monovalent cations. Metal porphyrins selectively bind to G-qs complexes to form DNAzyme, which can exhibit peroxidase and other catalytic activity simila...

  15. The high-resolution crystal structure of a parallel intermolecular DNA G-4 quadruplex/drug complex employing syn glycosyl linkages.

    Science.gov (United States)

    Clark, George R; Pytel, Patrycja D; Squire, Christopher J

    2012-07-01

    We have determined the X-ray structure of the complex between the DNA quadruplex d(5'-GGGG-3')(4) and daunomycin, as a potential model for studying drug-telomere interactions. The structure was solved at 1.08 Å by direct methods in space group I4. The asymmetric unit comprises a linear arrangement of one d(GGGG) strand, four daunomycin molecules, a second d(GGGG) strand facing in the opposite direction to the first, and Na and Mg cations. The crystallographic 4-fold axis generates the biological unit, which is a 12-layered structure comprising two sets of four guanine layers, with four layers each of four daunomycins stacked between the 5' faces of the two quadruplexes. The daunomycin layers fall into two groups which are novel in their mode of self assembly. The only contacts between daunomycin molecules within any one of these layers are van der Waals interactions, however there is substantial π-π stacking between successive daunomycin layers and also with adjacent guanine layers. The structure differs significantly from all other parallel d(TGGGGT)(4) quadruplexes in that the 5' guanine adopts the unusual syn glycosyl linkage, refuting the widespread belief that such conformations should all be anti. In contrast to the related d(TGGGGT)/daunomycin complex, there are no ligand-quadruplex groove insertion interactions.

  16. Label-free ultrasensitive detection of telomerase activity via multiple telomeric hemin/G-quadruplex triggered polyaniline deposition and a DNA tetrahedron-structure regulated signal.

    Science.gov (United States)

    Liu, Yuanjian; Wei, Min; Liu, Xu; Wei, Wei; Zhao, Hongyu; Zhang, Yuanjian; Liu, Songqin

    2016-01-31

    Label-free detection of telomerase activity was done by using telomeric hemin/G-quadruplex triggered polyaniline deposition, not only on themselves but also on the DNA tetrahedron-structure (DTS). DTS size has a great impact on telomerase accessibility, reactivity and detection sensitivity. The method has been used to evaluate bladder cancer development.

  17. Voltammetry and Molecular Assembly of G-quadruplex DNAzyme on Single-crystal Au(111)-electrode Surfaces – Hemin as an Electrochemical Intercalator

    DEFF Research Database (Denmark)

    Zhang, Ling; Ulstrup, Jens; Zhang, Jingdong

    2016-01-01

    DNA quadruplexes (qs’s) are a class of “non-canonical” oligonucleotides (OGNs) composed of stacked guanine (G) quartets generally stabilized by monovalent cations. Metal porphyrins selectively bind to G-qs complexes to form DNAzyme, which can exhibit peroxidase and other catalytic activity simila...

  18. Is the P3 amplitude reduction seen in externalizing psychopathology attributable to stimulus sequence effects?

    Science.gov (United States)

    Gilmore, Casey S; Malone, Stephen M; Iacono, William G

    2012-02-01

    P3 amplitude reduction (P3-AR) is associated with biological vulnerability to a spectrum of externalizing (EXT) disorders, such as conduct disorder, antisocial behavior, and substance use disorders. P3 amplitude, however, can be affected by the context within which it is measured, for example, by the position of the target in the sequence of stimuli during an oddball task. We hypothesized that EXT-related P3-AR may be due to attention or working memory deficits in EXT that would weaken these stimulus sequence effects. Using a community-based sample of adolescent males, we examined the relationship between P3 and EXT as a function of the number of standards preceding the target. Higher EXT was associated with significantly smaller P3 amplitude, regardless of the number of standards preceding the target. These results suggest that P3-AR in EXT does not vary as a function of stimulus sequence, further supporting P3-AR as an endophenotype for EXT disorders.

  19. Effect of single-point sequence alterations on the aggregationpropensity of a model protein

    Energy Technology Data Exchange (ETDEWEB)

    Bratko, Dusan; Cellmer, Troy; Prausnitz, John M.; Blanch, Harvey W.

    2005-10-07

    Sequences of contemporary proteins are believed to have evolved through process that optimized their overall fitness including their resistance to deleterious aggregation. Biotechnological processing may expose therapeutic proteins to conditions that are much more conducive to aggregation than those encountered in a cellular environment. An important task of protein engineering is to identify alternative sequences that would protect proteins when processed at high concentrations without altering their native structure associated with specific biological function. Our computational studies exploit parallel tempering simulations of coarse-grained model proteins to demonstrate that isolated amino-acid residue substitutions can result in significant changes in the aggregation resistance of the protein in a crowded environment while retaining protein structure in isolation. A thermodynamic analysis of protein clusters subject to competing processes of folding and association shows that moderate mutations can produce effects similar to those caused by changes in system conditions, including temperature, concentration, and solvent composition that affect the aggregation propensity. The range of conditions where a protein can resist aggregation can therefore be tuned by sequence alterations although the protein generally may retain its generic ability for aggregation.

  20. Main-Sequence Effective Temperatures from a Revised Mass-Luminosity Relation Based on Accurate Properties

    CERN Document Server

    Eker, Z; Soydugan, E; Bilir, S; Gokce, E Yaz; Steer, I; Tuysuz, M; Senyuz, T; Demircan, O

    2015-01-01

    The mass-luminosity (M-L), mass-radius (M-R) and mass-effective temperature ($M-T_{eff}$) diagrams for a subset of galactic nearby main-sequence stars with masses and radii accurate to $\\leq 3\\%$ and luminosities accurate to $\\leq 30\\%$ (268 stars) has led to a putative discovery. Four distinct mass domains have been identified, which we have tentatively associated with low, intermediate, high, and very high mass main-sequence stars, but which nevertheless are clearly separated by three distinct break points at 1.05, 2.4, and 7$M_{\\odot}$ within the mass range studied of $0.38-32M_{\\odot}$. Further, a revised mass-luminosity relation (MLR) is found based on linear fits for each of the mass domains identified. The revised, mass-domain based MLRs, which are classical ($L \\propto M^{\\alpha}$), are shown to be preferable to a single linear, quadratic or cubic equation representing as an alternative MLR. Stellar radius evolution within the main-sequence for stars with $M>1M_{\\odot}$ is clearly evident on the M-R d...

  1. EFFECTS OF 4-CHLOROPHENOL LOADINGS ON ACCLIMATION OF BIOMASS WITH OPTIMIZED FIXED TIME SEQUENCING BATCH REACTOR

    Directory of Open Access Journals (Sweden)

    H. Movahedyan, A. Assadi, M. M. Amin

    2008-10-01

    Full Text Available Abstract: Chlorinated phenols in many industrial effluents are usually difficult to be removed by conventional biological treatment processes. Performance of the aerobic sequencing batch reactor treating 4-chlorophenol containing wastewater at different loadings rates from 0.0075 to 1.2 g4CP/L.d was evaluated. The sequencing batch reactor was operated with fill, react, settle and decant phases in the order of 10:370:90:10 min, respectively, for a cycle time of 8 h at 10 days solid retention time and 16 h hydraulic retention time in the stable period. The effects of 4-chlorophenol loadings on the 4-chlorophenol and chemical oxygen demand removal percents, yield coefficient (Y, biomass variation and sludge volume index were investigated. High chemical oxygen demand removal efficiencies (95±3.5% and approximately complete 4-chlorophenol removal (>99% were observed even in the absence of growth substrate. The degradation of 4-chlorophenol led to formation of 5-chloro-2-hydroxymuconic semialdehyde, which was more oxidized, indicating complete disappearance of 4-chlorophenol via meta-cleavage pathway. A compact sludge with excellent settleability (sludge volume index=47±6.1 mL/g developed during entire acclimation period. High removal efficiencies with sequencing batch reactor may be due to enforced short term unsteady state conditions coupled with periodic exposure of the microorganisms to defined process conditions which facilitate the required metabolic pathways for treating xenobiotics containing wastewater.

  2. The Effect of Interference on Temporal Order Memory for Random and Fixed Sequences in Nondemented Older Adults

    Science.gov (United States)

    Tolentino, Jerlyn C.; Pirogovsky, Eva; Luu, Trinh; Toner, Chelsea K.; Gilbert, Paul E.

    2012-01-01

    Two experiments tested the effect of temporal interference on order memory for fixed and random sequences in young adults and nondemented older adults. The results demonstrate that temporal order memory for fixed and random sequences is impaired in nondemented older adults, particularly when temporal interference is high. However, temporal order…

  3. The Effects of Meiosis/Genetics Integration and Instructional Sequence on College Biology Student Achievement in Genetics.

    Science.gov (United States)

    Browning, Mark

    The purpose of the research was to manipulate two aspects of genetics instruction in order to measure their effects on college, introductory biology students' achievement in genetics. One instructional sequence that was used dealt first with monohybrid autosomal inheritance patterns, then sex-linkage. The alternate sequence was the reverse.…

  4. A single molecule study of G-quadruplex and short duplex DNA structures

    Science.gov (United States)

    Roy, William A., Jr.

    Given that certain conditions are met, a single stranded DNA/RNA (ssDNA/RNA) structure called G-quadruplex (GQ) can form in regions throughout the genome, including at the telomeres and internal regions of the chromosomes. These structures serve various functions depending on the region in which they form which include protecting the chromosome ends, interfering with telomere elongation in cancer cells, and regulating transcription and translation level gene expression. Due to their high stability, various cellular mechanisms, such as GQ destabilizing proteins, are employed to unfold these structures during DNA replication or repair. Yet, their distinct layered structure has made GQs an attractive drug target in cancer treatment as GQ stabilizing molecules could inhibit telomerase dependent telomere elongation, a mechanism occurring in the majority of cancer cells to avoid senescence and apoptosis. However, proteins or small molecules interact with GQ that is under the influence of various cellular tension mechanisms, including the tension applied by other nearby molecules or the tension due to DNA structure within the chromatin context. Therefore, it is important to characterize the stability of various GQs and their response to interacting molecules when subjected to a tensile force. We employed a novel DNA-based nano tension generator that utilizes the elastic properties of circularized short double-stranded DNA (dsDNA) oligonucleotides to apply tension on the GQ. Since this is a completely new approach, the majority of this thesis was dedicated to proof-of-principle studies that demonstrated the feasibility and functionality of the method.

  5. Ligand binding to telomeric G-quadruplex DNA investigated by funnel-metadynamics simulations

    Science.gov (United States)

    Moraca, Federica; Amato, Jussara; Ortuso, Francesco; Artese, Anna; Novellino, Ettore; Alcaro, Stefano; Parrinello, Michele; Limongelli, Vittorio

    2017-01-01

    G-quadruplexes (G4s) are higher-order DNA structures typically present at promoter regions of genes and telomeres. Here, the G4 formation decreases the replicative DNA at each cell cycle, finally leading to apoptosis. The ability to control this mitotic clock, particularly in cancer cells, is fascinating and passes through a rational understanding of the ligand/G4 interaction. We demonstrate that an accurate description of the ligand/G4 binding mechanism is possible using an innovative free-energy method called funnel-metadynamics (FM), which we have recently developed to investigate ligand/protein interaction. Using FM simulations, we have elucidated the binding mechanism of the anticancer alkaloid berberine to the human telomeric G4 (d[AG3(T2AG3)3]), computing also the binding free-energy landscape. Two ligand binding modes have been identified as the lowest energy states. Furthermore, we have found prebinding sites, which are preparatory to reach the final binding mode. In our simulations, the ions and the water molecules have been explicitly represented and the energetic contribution of the solvent during ligand binding evaluated. Our theoretical results provide an accurate estimate of the absolute ligand/DNA binding free energy (ΔGb0 = −10.3 ± 0.5 kcal/mol) that we validated through steady-state fluorescence binding assays. The good agreement between the theoretical and experimental value demonstrates that FM is a most powerful method to investigate ligand/DNA interaction and can be a useful tool for the rational design also of G4 ligands. PMID:28232513

  6. Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro

    Indian Academy of Sciences (India)

    Swathi Kota; Hari S Misra

    2015-12-01

    Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase lb of D. radiodurans (DraTopolB) could change the electrophoretic mobility of fast migrating intramolecular rec-G4 DNA into the slow migrating species. DraTopolB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular rec-G4 DNA structures stabilized by K+. On the contrary, when DraTopolB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopolB. This implies that DraTopolB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopolB activity on intramolecular G4 DNA structures. These results suggested that DraTopolB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.

  7. Probing the effect of promoters on noise in gene expression using thousands of designed sequences.

    Science.gov (United States)

    Sharon, Eilon; van Dijk, David; Kalma, Yael; Keren, Leeat; Manor, Ohad; Yakhini, Zohar; Segal, Eran

    2014-10-01

    Genetically identical cells exhibit large variability (noise) in gene expression, with important consequences for cellular function. Although the amount of noise decreases with and is thus partly determined by the mean expression level, the extent to which different promoter sequences can deviate away from this trend is not fully known. Here, we present a high-throughput method for measuring promoter-driven noise for thousands of designed synthetic promoters in parallel. We use it to investigate how promoters encode different noise levels and find that the noise levels of promoters with similar mean expression levels can vary more than one order of magnitude, with nucleosome-disfavoring sequences resulting in lower noise and more transcription factor binding sites resulting in higher noise. We propose a kinetic model of gene expression that takes into account the nonspecific DNA binding and one-dimensional sliding along the DNA, which occurs when transcription factors search for their target sites. We show that this assumption can improve the prediction of the mean-independent component of expression noise for our designed promoter sequences, suggesting that a transcription factor target search may affect gene expression noise. Consistent with our findings in designed promoters, we find that binding-site multiplicity in native promoters is associated with higher expression noise. Overall, our results demonstrate that small changes in promoter DNA sequence can tune noise levels in a manner that is predictable and partly decoupled from effects on the mean expression levels. These insights may assist in designing promoters with desired noise levels.

  8. Screening of a chemical library by HT-G4-FID for discovery of selective G-quadruplex binders.

    Science.gov (United States)

    Largy, Eric; Saettel, Nicolas; Hamon, Florian; Dubruille, Sylvie; Teulade-Fichou, Marie-Paule

    2012-01-01

    Due to the lack of structural guidelines about G-quadruplex ligands, rational design cannot be the only approach to discover potent G4-ligands. As a complementary approach, screening of chemical library may provide interesting scaffolds known as hits provided that specific tools are available. In this work, the Institut Curie-CNRS chemical library was firstly screened by chemoinformatics methods. Similarity estimations by comparison with reference compounds (Phen-DC3, 360A, MMQ12) provided a set of molecules, which were then evaluated by high-throughput G4-FID (HT-G4-FID) against various G-quadruplex DNA. A full investigation of the most interesting molecules, using the HT-G4-FID assay and molecular modeling, supplied an interesting structure-activity relationship confirming the efficiency of this general approach. Overall, we demonstrated that HT-G4-FID coupled with screening of chemical libraries is a powerful tool to identify new G4-DNA binding scaffolds.

  9. Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

    Science.gov (United States)

    Du, Yi-Chen; Zhu, Li-Na; Kong, De-Ming

    2016-12-15

    To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design.

  10. Insights into how nucleotide supplements enhance the peroxidase-mimicking DNAzyme activity of the G-quadruplex/hemin system.

    Science.gov (United States)

    Stefan, Loic; Denat, Franck; Monchaud, David

    2012-09-01

    Since the initial discovery of the catalytic capability of short DNA fragments, this peculiar enzyme-like property (termed DNAzyme) has continued to garner much interest in the scientific community because of the virtually unlimited applications in developing new molecular devices. Alongside the exponential rise in the number of DNAzyme applications in the last past years, the search for convenient ways to improve its overall efficiency has only started to emerge. Credence has been lent to this strategy by the recent demonstration that the quadruplex-based DNAzyme proficiency can be enhanced by ATP supplements. Herein, we have made a further leap along this path, trying first of all to decipher the actual DNAzyme catalytic cycle (to gain insights into the steps ATP may influence), and subsequently investigating in detail the influence of all the parameters that govern the catalytic efficiency. We have extended this study to other nucleotides and quadruplexes, thus demonstrating the versatility and broad applicability of such an approach. The defined exquisitely efficient DNAzyme protocols were exploited to highlight the enticing advantages of this method via a 96-well plate experiment that enables the detection of nanomolar DNA concentrations in real-time with the naked-eye (see movie as Supplementary Data).

  11. Structural dynamics of possible late-stage intermediates in folding of quadruplex DNA studied by molecular simulations

    Science.gov (United States)

    Stadlbauer, Petr; Krepl, Miroslav; Cheatham, Thomas E.; Koča, Jaroslav; Šponer, Jiří

    2013-01-01

    Explicit solvent molecular dynamics simulations have been used to complement preceding experimental and computational studies of folding of guanine quadruplexes (G-DNA). We initiate early stages of unfolding of several G-DNAs by simulating them under no-salt conditions and then try to fold them back using standard excess salt simulations. There is a significant difference between G-DNAs with all-anti parallel stranded stems and those with stems containing mixtures of syn and anti guanosines. The most natural rearrangement for all-anti stems is a vertical mutual slippage of the strands. This leads to stems with reduced numbers of tetrads during unfolding and a reduction of strand slippage during refolding. The presence of syn nucleotides prevents mutual strand slippage; therefore, the antiparallel and hybrid quadruplexes initiate unfolding via separation of the individual strands. The simulations confirm the capability of G-DNA molecules to adopt numerous stable locally and globally misfolded structures. The key point for a proper individual folding attempt appears to be correct prior distribution of syn and anti nucleotides in all four G-strands. The results suggest that at the level of individual molecules, G-DNA folding is an extremely multi-pathway process that is slowed by numerous misfolding arrangements stabilized on highly variable timescales. PMID:23700306

  12. Label-free fluorescent sensor for lead ion detection based on lead(II)-stabilized G-quadruplex formation.

    Science.gov (United States)

    Zhan, Shenshan; Wu, Yuangen; Luo, Yanfang; Liu, Le; He, Lan; Xing, Haibo; Zhou, Pei

    2014-10-01

    A label-free fluorescent DNA sensor for the detection of lead ions (Pb(2+)) based on lead(II)-stabilized G-quadruplex formation is proposed in this article. A guanine (G)-rich oligonucleotide, T30695, was used as a recognition probe, and a DNA intercalator, SYBR Green I (SG), was used as a signal reporter. In the absence of Pb(2+), the SG intercalated with the single-stranded random-coil T30695 and emitted strong fluorescence. While in the presence of Pb(2+), the random-coil T30695 would fold into a G-quadruplex structure and the SG could barely show weak fluorescence, and the fluorescence intensity was inversely proportional to the involving amount of Pb(2+). Based on this, a selective lead ion sensor with a limit of detection of 3.79 ppb (parts per billion) and a detection range from 0 to 600 ppb was constructed. Because detection for real samples was also demonstrated to be reliable, this simple, low-cost, sensitive, and selective sensor holds good potential for Pb(2+) detection in real environmental samples.

  13. Daytime sleep has no effect on the time course of motor sequence and visuomotor adaptation learning.

    Science.gov (United States)

    Backhaus, Winifried; Braaß, Hanna; Renné, Thomas; Krüger, Christian; Gerloff, Christian; Hummel, Friedhelm C

    2016-05-01

    Sleep has previously been claimed to be essential for the continued learning processes of declarative information as well as procedural learning. This study was conducted to examine the importance of sleep, especially the effects of midday naps, on motor sequence and visuomotor adaptation learning. Thirty-five (27 females) healthy, young adults aged between 18 and 30years of age participated in the current study. Addressing potential differences in explicit sequence and motor adaptation learning participants were asked to learn both, a nine-element explicit sequence and a motor adaptation task, in a crossover fashion on two consecutive days. Both tasks were performed with their non-dominant left hand. Prior to learning, each participant was randomized to one of three interventions; (1) power nap: 10-20min sleep, (2) long nap: 50-80min sleep or (3) a 45-min wake-condition. Performance of the motor learning task took place prior to and after a midday rest period, as well as after a night of sleep. Both sleep conditions were dominated by Stage N2 sleep with embedded sleep spindles, which have been described to be associated with enhancement of motor performance. Significant performance changes were observed in both tasks across all interventions (sleep and wake) confirming that learning took place. In the present setup, the magnitude of motor learning was not sleep-dependent in young adults - no differences between the intervention groups (short nap, long nap, no nap) could be found. The effect of the following night of sleep was not influenced by the previous midday rest or sleep period. This finding may be related to the selectiveness of the human brain enhancing especially memory being thought of as important in the future. Previous findings on motor learning enhancing effects of sleep, especially of daytime sleep, are challenged.

  14. A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Shunbi, E-mail: xieshunbi@163.com; Chai, Yaqin, E-mail: yaqinchai@swu.edu.cn; Yuan, Yali; Bai, Lijuan; Yuan, Ruo, E-mail: yuanruo@swu.edu.cn

    2014-06-01

    Highlights: • This assay is label-free, the signal can be read out by measuring the electrochemical signal of hemin. • The hemin/G-quadruplex HRP-DNAzyme nanowires were formed via EXPAR reaction and HCR. • The prepared aptasensor exhibited low detection limit and wide linear range to TB. - Abstract: In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR’s ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H{sub 2}O{sub 2}. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1 pM–50 nM with a detection limit of 33 fM (defined as S/N = 3) for TB.

  15. Sensitive pseudobienzyme electrocatalytic DNA biosensor for mercury(II) ion by using the autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Yali [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); College of resources and environments, Southwest University, Chongqing 400715 (China); Gao, Min [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Liu, Guangpeng [College of resources and environments, Southwest University, Chongqing 400715 (China); Chai, Yaqin [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Wei, Shiqing, E-mail: sqwei@swu.edu.cn [College of resources and environments, Southwest University, Chongqing 400715 (China); Yuan, Ruo, E-mail: yuanruo@swu.edu.cn [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

    2014-02-06

    Graphical abstract: -- Highlights: •An ultrasensitive detection system for Hg{sup 2+} detection was presented. •The autonomously assembled hemin/G-quadruplex DNAzyme nanowires were employed. •The DNAzyme simultaneously served as an NADH oxidase and HRP-mimicking DNAzyme. •The DNAzyme nanowires served as carrier for loading substantial redox probe Thi. -- Abstract: Herein, a novel sensitive pseudobienzyme electrocatalytic DNA biosensor was proposed for mercury ion (Hg{sup 2+}) detection by using autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification. Thiol functionalized capture DNA was firstly immobilized on a nano-Au modified glass carbon electrode (GCE). In presence of Hg{sup 2+}, the specific coordination between Hg{sup 2+} and T could result in the assembly of primer DNA on the electrode, which successfully triggered the HCR to form the hemin/G-quadruplex DNAzyme nanowires with substantial redox probe thionine (Thi). In the electrolyte of PBS containing NADH, the hemin/G-quadruplex nanowires firstly acted as an NADH oxidase to assist the concomitant formation of H{sub 2}O{sub 2} in the presence of dissolved O{sub 2}. Then, with the redox probe Thi as electron mediator, the hemin/G-quadruplex nanowires acted as an HRP-mimicking DNAzyme that quickly bioelectrocatalyzed the reduction of produced H{sub 2}O{sub 2}, which finally led to a dramatically amplified electrochemical signal. This method has demonstrated a high sensitivity of Hg{sup 2+} detection with the dynamic concentration range spanning from 1.0 ng L{sup −1} to 10 mg L{sup −1} Hg{sup 2+} and a detection limit of 0.5 ng L{sup −1} (2.5 pM) at the 3S{sub blank} level, and it also demonstrated excellent selectivity against other interferential metal ions.

  16. Sequence-non-specific effects generated by various types of RNA interference triggers.

    Science.gov (United States)

    Olejniczak, Marta; Urbanek, Martyna O; Jaworska, Edyta; Witucki, Lukasz; Szczesniak, Michal W; Makalowska, Izabela; Krzyzosiak, Wlodzimierz J

    2016-02-01

    RNA interference triggers such as short interfering RNA (siRNA) or genetically encoded short hairpin RNA (shRNA) and artificial miRNA (sh-miR) are widely used to silence the expression of specific genes. In addition to silencing selected targets, RNAi reagents may induce various side effects, including immune responses. To determine the molecular markers of immune response activation when using RNAi reagents, we analyzed the results of experiments gathered in the RNAimmuno (v 2.0) and GEO Profiles databases. To better characterize and compare cellular responses to various RNAi reagents in one experimental system, we designed a reagent series in corresponding siRNA, D-siRNA, shRNA and sh-miR forms. To exclude sequence-specific effects the reagents targeted 3 different transcripts (Luc, ATXN3 and HTT). We demonstrate that RNAi reagents induce a broad variety of sequence-non-specific effects, including the deregulation of cellular miRNA levels. Typical siRNAs are weak stimulators of interferon response but may saturate the miRNA biogenesis pathway, leading to the downregulation of highly expressed miRNAs, whereas plasmid-based reagents induce known markers of immune response and may alter miRNA levels and their isomiR composition.

  17. Dissecting the roles of local packing density and longer-range effects in protein sequence evolution.

    Science.gov (United States)

    Shahmoradi, Amir; Wilke, Claus O

    2016-06-01

    What are the structural determinants of protein sequence evolution? A number of site-specific structural characteristics have been proposed, most of which are broadly related to either the density of contacts or the solvent accessibility of individual residues. Most importantly, there has been disagreement in the literature over the relative importance of solvent accessibility and local packing density for explaining site-specific sequence variability in proteins. We show that this discussion has been confounded by the definition of local packing density. The most commonly used measures of local packing, such as contact number and the weighted contact number, represent the combined effects of local packing density and longer-range effects. As an alternative, we propose a truly local measure of packing density around a single residue, based on the Voronoi cell volume. We show that the Voronoi cell volume, when calculated relative to the geometric center of amino-acid side chains, behaves nearly identically to the relative solvent accessibility, and each individually can explain, on average, approximately 34% of the site-specific variation in evolutionary rate in a data set of 209 enzymes. An additional 10% of variation can be explained by nonlocal effects that are captured in the weighted contact number. Consequently, evolutionary variation at a site is determined by the combined effects of the immediate amino-acid neighbors of that site and effects mediated by more distant amino acids. We conclude that instead of contrasting solvent accessibility and local packing density, future research should emphasize on the relative importance of immediate contacts and longer-range effects on evolutionary variation. Proteins 2016; 84:841-854. © 2016 Wiley Periodicals, Inc.

  18. Molecular simulations of polycation-DNA binding exploring the effect of peptide chemistry and sequence in nuclear localization sequence based polycations.

    Science.gov (United States)

    Elder, Robert M; Jayaraman, Arthi

    2013-10-10

    Gene therapy relies on the delivery of DNA into cells, and polycations are one class of vectors enabling efficient DNA delivery. Nuclear localization sequences (NLS), cationic oligopeptides that target molecules for nuclear entry, can be incorporated into polycations to improve their gene delivery efficiency. We use simulations to study the effect of peptide chemistry and sequence on the DNA-binding behavior of NLS-grafted polycations by systematically mutating the residues in the grafts, which are based on the SV40 NLS (peptide sequence PKKKRKV). Replacing arginine (R) with lysine (K) reduces binding strength by eliminating arginine-DNA interactions, but placing R in a less hindered location (e.g., farther from the grafting point to the polycation backbone) has surprisingly little effect on polycation-DNA binding strength. Changing the positions of the hydrophobic proline (P) and valine (V) residues relative to the polycation backbone changes hydrophobic aggregation within the polycation and, consequently, changes the conformational entropy loss that occurs upon polycation-DNA binding. Since conformational entropy loss affects the free energy of binding, the positions of P and V in the grafts affect DNA binding affinity. The insight from this work guides synthesis of polycations with tailored DNA binding affinity and, in turn, efficient DNA delivery.

  19. Effects of studying sequences of process-oriented and product-oriented worked examples on troubleshooting transfer efficiency

    NARCIS (Netherlands)

    Van Gog, Tamara; Paas, Fred; Van Merriënboer, Jeroen

    2008-01-01

    Van Gog, T., Paas, F., & Van Merriënboer, J. J. G. (2008). Effects of studying sequences of process-oriented and product-oriented worked examples on troubleshooting transfer efficiency. Learning and Instruction, 18, 211-222.

  20. Effects of GC bias in next-generation-sequencing data on de novo genome assembly.

    Science.gov (United States)

    Chen, Yen-Chun; Liu, Tsunglin; Yu, Chun-Hui; Chiang, Tzen-Yuh; Hwang, Chi-Chuan

    2013-01-01

    Next-generation-sequencing (NGS) has revolutionized the field of genome assembly because of its much higher data throughput and much lower cost compared with traditional Sanger sequencing. However, NGS poses new computational challenges to de novo genome assembly. Among the challenges, GC bias in NGS data is known to aggravate genome assembly. However, it is not clear to what extent GC bias affects genome assembly in general. In this work, we conduct a systematic analysis on the effects of GC bias on genome assembly. Our analyses reveal that GC bias only lowers assembly completeness when the degree of GC bias is above a threshold. At a strong GC bias, the assembly fragmentation due to GC bias can be explained by the low coverage of reads in the GC-poor or GC-rich regions of a genome. This effect is observed for all the assemblers under study. Increasing the total amount of NGS data thus rescues the assembly fragmentation because of GC bias. However, the amount of data needed for a full rescue depends on the distribution of GC contents. Both low and high coverage depths due to GC bias lower the accuracy of assembly. These pieces of information provide guidance toward a better de novo genome assembly in the presence of GC bias.

  1. Competitive binding exchange between alkali metal ions (K+, Rb+, and Cs+) and Na+ ions bound to the dimeric quadruplex [d(G4T4G4)]2: a 23Na and 1H NMR study.

    Science.gov (United States)

    Cesare Marincola, Flaminia; Virno, Ada; Randazzo, Antonio; Mocci, Francesca; Saba, Giuseppe; Lai, Adolfo

    2009-12-01

    A comparative study of the competitive cation exchange between the alkali metal ions K+, Rb+, and Cs+ and the Na+ ions bound to the dimeric quadruplex [d(G4T4G4)]2 was performed in aqueous solution by a combined use of the 23Na and 1H NMR spectroscopy. The titration data confirm the different binding affinities of these ions for the G-quadruplex and, in particular, major differences in the behavior of Cs+ as compared to the other ions were found. Accordingly, Cs+ competes with Na+ only for the binding sites at the quadruplex surface (primarily phosphate groups), while K+ and Rb+ are also able to replace sodium ions located inside the quadruplex. Furthermore, the 1H NMR results relative to the CsCl titration evidence a close approach of Cs+ ions to the phosphate groups in the narrow groove of [d(G4T4G4)]2. Based on a three-site exchange model, the 23Na NMR relaxation data lead to an estimate of the relative binding affinity of Cs+ versus Na+ for the quadruplex surface of 0.5 at 298 K. Comparing this value to those reported in the literature for the surface of the G-quadruplex formed by 5'-guanosinemonophosphate and for the surface of double-helical DNA suggests that topology factors may have an important influence on the cation affinity for the phosphate groups on DNA.

  2. Antitumor polycyclic acridines. 20. Search for DNA quadruplex binding selectivity in a series of 8,13-dimethylquino[4,3,2-kl]acridinium salts: telomere-targeted agents.

    Science.gov (United States)

    Cheng, Mai-Kim; Modi, Chetna; Cookson, Jennifer C; Hutchinson, Ian; Heald, Robert A; McCarroll, Andrew J; Missailidis, Sotiris; Tanious, Farial; Wilson, W David; Mergny, Jean-Louis; Laughton, Charles A; Stevens, Malcolm F G

    2008-02-28

    The growth-inhibitory activities of an extensive series of quaternized quino[4,3,2- kl]acridinium salts against tumor cell lines in vitro have been measured and their biological properties interpreted in the light of differential binding to different DNA isoforms. Selectivity for quadruplex DNA binding and stabilization by compounds were explored through an array of methods: UV absorption and fluorescence emission spectroscopy, surface plasmon resonance, and competition dialysis. Quadruplex DNA interaction was further characterized through FRET and DNA polymerase arrest assays. Telomerase inhibition, inferred from the TRAP assay, is attributed to quadruplex stabilization, supported by the strong correlation (R(2) = 0.81) across the series between quadruplex DNA binding affinity and TRAP inhibition potency. Growth inhibition potency in the NCI60 human tumor cell line panel is more marked in compounds with greater DNA duplex binding affinity (R(2) = 0.82). Quantification of relative quadruplex and duplex binding affinity constants puts some of these ligands among the most selective quadruplex DNA interactive agents reported to date.

  3. Effects of Aftershock Declustering in Risk Modeling: Case Study of a Subduction Sequence in Mexico

    Science.gov (United States)

    Kane, D. L.; Nyst, M.

    2014-12-01

    Earthquake hazard and risk models often assume that earthquake rates can be represented by a stationary Poisson process, and that aftershocks observed in historical seismicity catalogs represent a deviation from stationarity that must be corrected before earthquake rates are estimated. Algorithms for classifying individual earthquakes as independent mainshocks or as aftershocks vary widely, and analysis of a single catalog can produce considerably different earthquake rates depending on the declustering method implemented. As these rates are propagated through hazard and risk models, the modeled results will vary due to the assumptions implied by these choices. In particular, the removal of large aftershocks following a mainshock may lead to an underestimation of the rate of damaging earthquakes and potential damage due to a large aftershock may be excluded from the model. We present a case study based on the 1907 - 1911 sequence of nine 6.9 Mexico in order to illustrate the variability in risk under various declustering approaches. Previous studies have suggested that subduction zone earthquakes in Mexico tend to occur in clusters, and this particular sequence includes events that would be labeled as aftershocks in some declustering approaches yet are large enough to produce significant damage. We model the ground motion for each event, determine damage ratios using modern exposure data, and then compare the variability in the modeled damage from using the full catalog or one of several declustered catalogs containing only "independent" events. We also consider the effects of progressive damage caused by each subsequent event and how this might increase or decrease the total losses expected from this sequence.

  4. THE EFFECT OF BLENDING SEQUENCE ON PHASE MORPHOLOGY OF NYLON 6/ABS/SMA BLENDS

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The preparation process-dependent phase morphology of blends composed of nylon 6 and acryionitrile-butadienestyrene(ABS)over a composition range of 30-70 wt% using a styrene-maleic anhydride(SMA)copolymer as the compatibilizing agent with a constant content(5phr)was investigated.The results of the scanning electron microscope (SEM)observation revealed that compared with the binary blends of nylon 6 and ABS,the existence of SMA caused a composition shift of phase inversion to a higher weight fraction of nylon 6 when ABS was blended with the preblended nylon 6/SMA blend,while the co-continuous structures could be observed over a considerably narrower composition range when nylon 6 was blended with the pre-blended ABS/SMA blend.An examination through dynamic mechanical analysis (DMA)tests confirmed the results obtained with SEM.It is found that near the phase inversion region a remarkable change in the dynamic storage modulus(G')and the loss tangent(tanδ)appears.Moreover,the influence of blending sequence on the size of dispersed particles has been probed for uncompatibilized and compatibilized blends of nylon 6 and ABS over a wide range of compositions below or beyond the phase inversion points.For the blends of ABS dispersed in a nylon 6 matrix,little discernible effects of blending sequence on particle size could be observed.Furthermore,there exists a significant difference in morphologies of the blends prepared by nylon 6 particles dispersing in a ABS matrix in cases of different blending sequences used.Some possible factors responsible for the above asymmetric behaviors have been proposed.

  5. Exploiting Polydopamine Nanospheres to DNA Computing: A Simple, Enzyme-Free and G-Quadruplex-Free DNA Parity Generator/Checker for Error Detection during Data Transmission.

    Science.gov (United States)

    Fan, Daoqing; Wang, Erkang; Dong, Shaojun

    2017-01-18

    Molecular logic devices with various functions play an indispensable role in molecular data transmission/processing. However, during any kinds of data transmission, a constant and unavoidable circumstance is the appearance of bit errors, which have serious effects on the regular logic computation. Fortunately, these errors can be detected via plugging a parity generator (pG) at the transmitting terminal and a parity checker (pC) at the receiving terminal. Herein, taking advantage of the efficient adsorption/quenching ability of polydopamine nanospheres toward fluorophore-labeled single-stranded DNA, we explored this biocompatible nanomaterial to DNA logic computation and constructed the first simple, enzyme-free, and G-quadruplex-free DNA pG/pC for error detection through data transmission. Besides, graphene oxide (GO) was innovatively introduced as the "corrective element" to perform the output-correction function of pC. All the erroneous outputs were corrected to normal conditions completely, ensuring the regular operation of later logic computing. The total operation of this non-G4 pG/pC system (error checking/output-correction) could be completed within 1 h (about (1)/3 of previous G4 platform) in a simpler and more efficient way. Notably, the odd pG/pC with analogous functions was also achieved through negative logic conversion to the fabricated even one. Furthermore, the same system could also perform three-input concatenated logic computation (XOR-INHIBIT), enriching the complexity of PDs-based logic computation.

  6. Effect of photoperiod on the feline adipose transcriptome as assessed by RNA sequencing.

    Science.gov (United States)

    Mori, Akihiro; Kappen, Kelly L; Dilger, Anna C; Swanson, Kelly S

    2014-07-03

    Photoperiod is known to cause physiological changes in seasonal mammals, including changes in body weight, physical activity, reproductive status, and adipose tissue gene expression in several species. The objective of this study was to determine the effects of day length on the adipose transcriptome of cats as assessed by RNA sequencing. Ten healthy adult neutered male domestic shorthair cats were used in a randomized crossover design study. During two 12-wk periods, cats were exposed to either short days (8 hr light:16 hr dark) or long days (16 hr light:8 hr dark). Cats were fed a commercial diet to maintain baseline body weight to avoid weight-related bias. Subcutaneous adipose biopsies were collected at wk 12 of each period for RNA isolation and sequencing. A total of 578 million sequences (28.9 million/sample) were generated by Illumina sequencing. A total of 170 mRNA transcripts were differentially expressed between short day- and long day-housed cats. 89 annotated transcripts were up-regulated by short days, while 24 annotated transcripts were down-regulated by short days. Another 57 un-annotated transcripts were also different between groups. Adipose tissue of short day-housed cats had greater expression of genes involved with cell growth and differentiation (e.g., myostatin; frizzled-related protein), cell development and structure (e.g., cytokeratins), and protein processing and ubiquitination (e.g., kelch-like proteins). In contrast, short day-housed cats had decreased expression of genes involved with immune function (e.g., plasminogen activator inhibitor 1; chemokine (C-C motif) ligand 2; C-C motif chemokine 5; T-cell activators), and altered expression of genes associated with carbohydrate and lipid metabolism. Collectively, these gene expression changes suggest that short day housing may promote adipogenesis, minimize inflammation and oxidative stress, and alter nutrient metabolism in feline adipose tissue, even when fed to maintain body weight

  7. Effects of aging and freezing/thawing sequence on quality attributes of bovine and

    Directory of Open Access Journals (Sweden)

    Hyun-Wook Kim

    2017-02-01

    Full Text Available Objective The effects of aging and freezing/thawing sequence on color, physicochemical, and enzymatic characteristics of two beef muscles (Mm. gluteus medius, GM and biceps femoris, BF were evaluated. Methods Beef muscles at 3 d postmortem were assigned to four different combinations of aging and freezing/thawing sequence as follows; aging at 2°C for 3 wk (A3, never-frozen control, freezing at −28°C for 2 wk then thawing (F2, frozen/thawed-only, aging at 2°C for 3 wk, freezing at −28°C for 2 wk then thawing (A3F2, and freezing at −28°C for 2 wk, thawing then further aging at 2°C for 3 wk (F2A3. Results No significant interactions between different aging/freezing/thawing treatments and muscle type on all measurements were found. Postmortem aging, regardless of aging/freezing/thawing sequence, had no impact on color stability of frozen/thawed beef muscles (p<0.05. F2A3 resulted in higher purge loss than F2 and A3F2 treatments (p<0.05. A3F2 and F2A3 treatments resulted in lower shear force of beef muscles compared to F2 (p<0.05. Although there was no significant difference in glutathione peroxidase (GSH-Px activity, F2A3 had the highest β-N-acetyl glucominidase (BNAG activity in purge, but the lowest BNAG activity in muscle (p<0.05. GM muscle exhibited higher total color changes and purge loss, and lower GSH-Px activity than BF muscle. Conclusion The results from this present study indicate that different combinations of aging/freezing/thawing sequence would result in considerable impacts on meat quality attributes, particularly thaw/purge loss and tenderness. Developing a novel freezing strategy combined with postmortem aging will be beneficial for the food/meat industry to maximize its positive impacts on tenderness, while minimizing thaw/purge loss of frozen/thawed meat.

  8. Steady state effects in a two-pulse diffusion-weighted sequence

    Energy Technology Data Exchange (ETDEWEB)

    Zubkov, Mikhail; Stait-Gardner, Timothy; Price, William S. [Nanoscale Organisation and Dynamics Group, School of Science and Health, University of Western Sydney, Sydney (Australia); Stilbs, Peter [Division of Applied Physical Chemistry, Department of Chemistry, KTH Royal Institute of Technology, SE-10044 Stockholm (Sweden)

    2015-04-21

    In conventional nuclear magnetic resonance (NMR) diffusion measurements a significant amount of experimental time is used up by magnetization recovery, serving to prevent the formation of the steady state, as in the latter case the manifestation of diffusion is modulated by multiple applications of the pulse sequence and conventional diffusion coefficient inference procedures are generally not applicable. Here, an analytical expression for diffusion-related effects in a two-pulse NMR experiment (e.g., pulsed-gradient spin echo) in the steady state mode (with repetition times less than the longitudinal relaxation time of the sample) is derived by employing a Fourier series expansion within the solution of the Bloch-Torrey equations. Considerations are given for the transition conditions between the full relaxation and the steady state experiment description. The diffusion coefficient of a polymer solution (polyethylene glycol) is measured by a two-pulse sequence in the full relaxation mode and for a range of repetition times, approaching the rapid steady state experiment. The precision of the fitting employing the presented steady state solution by far exceeds that of the conventional fitting. Additionally, numerical simulations are performed yielding results strongly supporting the proposed description of the NMR diffusion measurements in the steady state.

  9. Facilitating and inhibiting effects of priming and selection criteria in a sequence of dichotic listening trials.

    Science.gov (United States)

    Saetrevik, Bjørn

    2012-08-01

    Competing models of attention make different predictions of how priming from recent stimulus processing could interact with intended selection. The present experiment examined the interaction between exogenous attention and endogenous priming across trial sequences. A sound cue directed attention to left, right or both sides before a dichotic syllable pair was presented. Participants were asked to report one syllable from each trial. Results showed that responses were slower on trials where one of the presented syllables had also been presented on the previous trial. Within these trials, the repeated syllable was selected less frequently, and the responses doing so were slower. Examined according to response choice on the preceding trial, syllables that had been ignored on the preceding trial tended to be ignored on the current trial (negative priming), while syllables that had been selected on the preceding trial tended to be selected on the current trial (positive priming). Responses that followed these selection biases were faster than responses that did not. Response selection was also influenced by the attention direction cue for the current trial, but not by the cue presented on the preceding trial. The results support an attentional model where traces from the preceding processing are retained, and current selection is biased to minimize cognitive conflict between recent and current processing. Negative priming appears to be due to after-effects of preceding processing, independently of the intentions behind that processing. The study accounts for positive and negative priming of dichotic listening sequences within an established, computationally viable biased competition framework.

  10. Effect of energy level sequences and neutron–proton interaction on α-particle preformation probability

    Energy Technology Data Exchange (ETDEWEB)

    Ismail, M.; Adel, A., E-mail: ahmedshosha200@yahoo.com

    2013-08-21

    A realistic density-dependent nucleon–nucleon (NN) interaction with finite-range exchange part which produces the nuclear matter saturation curve and the energy dependence of the nucleon–nucleus optical model potential is used to calculate the preformation probability, S{sub α}, of α-decay from different isotones with neutron numbers N=124,126,128,130 and 132. We studied the variation of S{sub α} with the proton number, Z, for each isotone and found the effect of neutron and proton energy levels of parent nuclei on the behavior of the α-particle preformation probability. We found that S{sub α} increases regularly with the proton number when the proton pair in α-particle is emitted from the same level and the neutron level sequence is not changed during the Z-variation. In this case the neutron–proton (n–p) interaction of the two levels, contributing to emission process, is too small. On the contrary, if the proton or neutron level sequence is changed during the emission process, S{sub α} behaves irregularly, the irregular behavior increases if both proton and neutron levels are changed. This behavior is accompanied by change or rapid increase in the strength of n–p interaction.

  11. The effect of starspots on the radii of low-mass pre-main sequence stars

    CERN Document Server

    Jackson, R J

    2014-01-01

    A polytropic model is used to investigate the effects of dark photospheric spots on the evolution and radii of magnetically active, low-mass (M<0.5Msun), pre-main sequence (PMS) stars. Spots slow the contraction along Hayashi tracks and inflate the radii of PMS stars by a factor of (1-beta)^{-N} compared to unspotted stars of the same luminosity, where beta is the equivalent covering fraction of dark starspots and N \\simeq 0.45+/-0.05. This is a much stronger inflation than predicted by the models of Spruit & Weiss (1986) for main sequence stars with the same beta, where N \\sim 0.2 to 0.3. These models have been compared to radii determined for very magnetically active K- and M-dwarfs in the young Pleiades and NGC 2516 clusters, and the radii of tidally-locked, low-mass eclipsing binary components. The binary components and ZAMS K-dwarfs have radii inflated by \\sim 10 per cent compared to an empirical radius-luminosity relation that is defined by magnetically inactive field dwarfs with interferometrica...

  12. Whole-genome sequencing reveals the effect of vaccination on the evolution of Bordetella pertussis.

    Science.gov (United States)

    Xu, Yinghua; Liu, Bin; Gröndahl-Yli-Hannuksila, Kirsi; Tan, Yajun; Feng, Lu; Kallonen, Teemu; Wang, Lichan; Peng, Ding; He, Qiushui; Wang, Lei; Zhang, Shumin

    2015-08-18

    Herd immunity can potentially induce a change of circulating viruses. However, it remains largely unknown that how bacterial pathogens adapt to vaccination. In this study, Bordetella pertussis, the causative agent of whooping cough, was selected as an example to explore possible effect of vaccination on the bacterial pathogen. We sequenced and analysed the complete genomes of 40 B. pertussis strains from Finland and China, as well as 11 previously sequenced strains from the Netherlands, where different vaccination strategies have been used over the past 50 years. The results showed that the molecular clock moved at different rates in these countries and in distinct periods, which suggested that evolution of the B. pertussis population was closely associated with the country vaccination coverage. Comparative whole-genome analyses indicated that evolution in this human-restricted pathogen was mainly characterised by ongoing genetic shift and gene loss. Furthermore, 116 SNPs were specifically detected in currently circulating ptxP3-containing strains. The finding might explain the successful emergence of this lineage and its spread worldwide. Collectively, our results suggest that the immune pressure of vaccination is one major driving force for the evolution of B. pertussis, which facilitates further exploration of the pathogenicity of B. pertussis.

  13. Effect of stacking sequence on the coefficients of mutual influence of composite laminates

    Science.gov (United States)

    Dupir (Hudișteanu, I.; Țăranu, N.; Axinte, A.

    2016-11-01

    Fiber reinforced polymeric (FRP) composites are nowadays widely used in engineering applications due to their outstanding features, such as high specific strength and specific stiffness as well as good corrosion resistance. A major advantage of fibrous polymeric composites is that their anisotropy can be controlled through suitable choice of the influencing parameters. The unidirectional fiber reinforced composites provide much higher longitudinal mechanical properties compared to the transverse ones. Therefore, composite laminates are formed by stacking two or more laminas, with different fiber orientations, as to respond to complex states of stresses. These laminates experience the effect of axial-shear coupling, which is caused by applying normal or shear stresses, implying shear or normal strains, respectively. The normal-shear coupling is expressed by the coefficients of mutual influence. They are engineering constants of primary interest for composite laminates, since the mismatch of the material properties between adjacent layers can produce interlaminar stresses and/or plies delamination. The paper presents the variation of the in-plane and flexural coefficients of mutual influence for three types of multi-layered composites, with different stacking sequences. The results are obtained using the Classical Lamination Theory (CLT) and are illustrated graphically in terms of fiber orientations, for asymmetric, antisymmetric and symmetric laminates. Conclusions are formulated on the variation of these coefficients, caused by the stacking sequence.

  14. Lowering the overall charge on TMPyP4 improves its selectivity for G-quadruplex DNA.

    Science.gov (United States)

    Ruan, Thomas L; Davis, Supriya J; Powell, Barrett M; Harbeck, Cole P; Habdas, Jan; Habdas, Piotr; Yatsunyk, Liliya A

    2017-01-01

    Ligands that stabilize non-canonical DNA structures called G-quadruplexes (GQs) might have applications in medicine as anti-cancer agents, due to the involvement of GQ DNA in a variety of cancer-related biological processes. Five derivatives of 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4), where a N-methylpyridyl group was replaced with phenyl (4P3), 4-aminophenyl (PN3M), 4-phenylamidoproline (PL3M), or 4-carboxyphenyl (PC3M and P2C2M) were investigated for their interactions with human telomeric DNA (Tel22) using fluorescence resonance energy transfer (FRET) assay, and UV-visible and circular dichroism spectroscopies in K(+) buffer. The molecules are cationic or zwitterionic with an overall charge of 3+ (4P3, PN3M, and PL3M), 2+ (PC3M) or neutral (P2C2M). All porphyrins except P2C2M stabilize human telomeric DNA in FRET assays by ∼20 °C at 5 eq CD melting experiments suggest that 4P3 is the most stabilizing ligand with a stabilization temperature of 16.8 °C at 4 eq. Importantly, 4P3, PC3M and PL3M demonstrate excellent selectivity for quadruplexes, far superior to that of TMPyP4. Binding constants, determined using UV-vis titrations, correlate with charge: triply cationic 4P3, PN3M and PL3M display Ka of 5-9 μM(-1), doubly cationic PC3M displays Ka of 1 μM(-1), and neutral P2C2M displays weak-to-no binding. UV-vis data suggest that binding interactions are driven by electrostatic attractions and that the binding mode may be base-stacking (or end-stacking) judging by the high values of red shift (15-20 nm) and hypochromicity (40-50%). We conclude that lowering the charge on TMPyP4 to 3+ can achieve the desired balance between stabilizing ability, affinity, and high selectivity required for an excellent quadruplex ligand.

  15. Interactome-wide prediction of protein-protein binding sites reveals effects of protein sequence variation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Felipe Leal Valentim

    Full Text Available The specificity of protein-protein interactions is encoded in those parts of the sequence that compose the binding interface. Therefore, understanding how changes in protein sequence influence interaction specificity, and possibly the phenotype, requires knowing the location of binding sites in those sequences. However, large-scale detection of protein interfaces remains a challenge. Here, we present a sequence- and interactome-based approach to mine interaction motifs from the recently published Arabidopsis thaliana interactome. The resultant proteome-wide predictions are available via www.ab.wur.nl/sliderbio and set the stage for further investigations of protein-protein binding sites. To assess our method, we first show that, by using a priori information calculated from protein sequences, such as evolutionary conservation and residue surface accessibility, we improve the performance of interface prediction compared to using only interactome data. Next, we present evidence for the functional importance of the predicted sites, which are under stronger selective pressure than the rest of protein sequence. We also observe a tendency for compensatory mutations in the binding sites of interacting proteins. Subsequently, we interrogated the interactome data to formulate testable hypotheses for the molecular mechanisms underlying effects of protein sequence mutations. Examples include proteins relevant for various developmental processes. Finally, we observed, by analysing pairs of paralogs, a correlation between functional divergence and sequence divergence in interaction sites. This analysis suggests that large-scale prediction of binding sites can cast light on evolutionary processes that shape protein-protein interaction networks.

  16. Microwave-assisted synthesis of ruthenium(II) complexes with alkynes as potential inhibitor by selectively recognizing c-myc G-quadruplex DNA.

    Science.gov (United States)

    Zhang, Shuangyan; Wu, Qiong; Zhang, Hao; Wang, Qi; Wang, Xicheng; Mei, Wenjie; Wu, Xiaohui; Zheng, Wenjie

    2017-11-01

    Herein, two polypyridyl ruthenium(II) complexes with alkynes, [Ru(bpy)2L](ClO4)2 (L=p-TEPIP (1) and p-BEPIP (2); bpy=2,2'-bipyridine; p-TEPIP=2-(4-trimethylsilylpropargyl)-1H-imidazo[4,5f][1,10]phenanthroline; p-BEPIP=2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline) have been successfully achieved in yields of 32%-89% by a Sonogashira coupling reaction under microwave irradiation. We studied these complexes as potential stabilizers of c-myc G-quadruplex DNA. Observations revealed that both complexes could selectively bind to and stabilize c-myc G-quadruplex DNA with a constant of approximately 1.61±0.78 and 9.47±4.20×10(3)M(-1), respectively, as determined from ITC (isothermal ttitration calorimetry) experiments, FRET (fluorescence resonance energy ttransfer) assay and competitive FRET assay. Moreover, the melting point (Tm) of the c-myc G-quadruplex DNA increased in the presence of 1 and 2 ([Ru]=0.2μM) by approximately 9 and 19.9°C, respectively. It is noteworthy that the conformation of the c-myc G-quadruplex DNA appeared to change when titrated with 1 and 2, which was accompanied by a negative-induced CD (circular dichroism) signal that appeared at a wavelength of 295nm. Furthermore, the conformational change in c-myc G-quadruplex DNA induced by 1 and 2have also been confirmed by TEM (transmission electron microscopy) and AFM (atomic force microscopy). Consequently, the replication of c-myc DNA was blocked by 1 and 2, and especially by 2, as verified by PCR (polymerase chain reaction) -stop assay and Western-blot assay. Thus, these ruthenium(II) complexes can be developed as potential inhibitors in chemotherapy through their binding and stabilization of c-myc G-quadruplex DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Phenyl 1,2,3-triazole-thymidine ligands stabilize G-quadruplex DNA, inhibit DNA synthesis and potentially reduce tumor cell proliferation over 3'-azido deoxythymidine.

    Science.gov (United States)

    Mahesh Kumar, Jerald; Idris, Mohammed M; Srinivas, Gunda; Vinay Kumar, Pallerla; Meghah, Vuppalapaty; Kavitha, Mitta; Reddy, Chada Raji; Mainkar, Prathama S; Pal, Biswajit; Chandrasekar, Srivari; Nagesh, Narayana

    2013-01-01

    Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4-L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2) and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC₅₀ value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). T(m) of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (K(d)≈10⁻⁷ M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC₅₀ values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are

  18. Phenyl 1,2,3-triazole-thymidine ligands stabilize G-quadruplex DNA, inhibit DNA synthesis and potentially reduce tumor cell proliferation over 3'-azido deoxythymidine.

    Directory of Open Access Journals (Sweden)

    Jerald Mahesh Kumar

    Full Text Available Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3 and non-nucleoside 1,2,3-triazoles (L4-L14 were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2 and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3, demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC₅₀ value for L3 was lowest (50 µM among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3 cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA than non quadruplex forming DNA (NQF DNA. T(m of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA. From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3 interact with quadruplex DNA with significantly higher affinity (K(d≈10⁻⁷ M. Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC₅₀ values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands

  19. What you learn is more than what you see: What can sequencing effects tell us about inductive category learning?

    Directory of Open Access Journals (Sweden)

    Paulo F. Carvalho

    2015-04-01

    Full Text Available Inductive category learning takes place across time. As such, it is not surprising that the sequence in which information is studied has an impact in what is learned and how efficient learning is. In this paper we review research on different learning sequences and how this impacts learning. We analyze different aspects of interleaved (frequent alternation between categories during study and blocked study (infrequent alternation between categories during study that might explain how and when one sequence of study results in improved learning. While these different sequences of study differ in the amount of temporal spacing and temporal juxtaposition between items of different categories, these aspects do not seem to account for the majority of the results available in the literature. However, differences in the type of category being studied and the duration of the retention interval between study and test may play an important role. We conclude that there is no single aspect that is able to account for all the evidence available. Understanding learning as a process of sequential comparisons in time and how different sequences fundamentally alter the statistics of this experience offers a promising framework for understanding sequencing effects in category learning. We use this framework to present novel predictions and hypotheses for future research on sequencing effects in inductive category learning.

  20. A New Semi-Empirical Technique For Computing Effective Temperatures For Main Sequence Stars From Their Mass And Radii

    Science.gov (United States)

    Aslan, Gürkan; Soydugan, Faruk; Eker, Zeki; Bilir, Selçuk; Bakış, Volkan

    2016-07-01

    A semi-empirical technique of improving effective temperature for main sequence stars from their observed mass and radius based on the Stefan-Boltzmann law, was introduced and applied to 450 main-sequence stars with accurate parameters. The method requires a mass-luminosity relation (MLR) and theoretical predictions of radius and effective temperature for stars at zero age main-sequence and at terminal age main-sequence. The MLRs, which act as if a catalyst, are necessary but have no effect on the final result. The present sample of main-sequence stars, which are members of the detached double-lined eclipsing binaries in the solar neighborhood chosen from Eker et al. (2014), have an error histogram for the observed effective temperatures with a peak at 2-3%. Errors of refined effective temperatures by the present method are the propagated errors of the observed masses and radii, that is, the refined temperatures and associated errors are independent of the observational temperatures and their associated errors. The histogram of the refined temperature errors shows a peak at less than 1%. A refined sample of stars (270 out of 450) with masses and radii accurate up to 3% and their refined effective temperatures has been used in this study to improve the classical MLRs. One may prefer, however, to use improved classical MLRs, which allows one to compute effective temperatures as accurate as 3.5%.

  1. Effect of Generalized Uncertainty Principle on Main-Sequence Stars and White Dwarfs

    CERN Document Server

    Moussa, Mohamed

    2015-01-01

    This paper addresses the effect of generalized uncertainty principle, emerged by a different approaches of quantum gravity within Planck scale, on thermodynamic properties of photon, non-relativistic ideal gases and degenerate fermions. A modification in pressure, particle number and energy density are calculated. Astrophysical objects such as main sequence stars and white dwarfs are examined and discussed as an application. A modification in Lane-Emden equation due to a change in a polytropic relation caused by the presence of quantum gravity, is investigated. The applicable range of quantum gravity parameters is estimated. The bounds in the perturbed parameters are relatively large but it may be considered reasonable values in the astrophysical regime.

  2. Genepleio Software for Effective Estimation of Gene Pleiotropy from Protein Sequences

    Directory of Open Access Journals (Sweden)

    Wenhai Chen

    2015-01-01

    Full Text Available Though pleiotropy, which refers to the phenomenon of a gene affecting multiple traits, has long played a central role in genetics, development, and evolution, estimation of the number of pleiotropy components remains a hard mission to accomplish. In this paper, we report a newly developed software package, Genepleio, to estimate the effective gene pleiotropy from phylogenetic analysis of protein sequences. Since this estimate can be interpreted as the minimum pleiotropy of a gene, it is used to play a role of reference for many empirical pleiotropy measures. This work would facilitate our understanding of how gene pleiotropy affects the pattern of genotype-phenotype map and the consequence of organismal evolution.

  3. Effect of Generalized Uncertainty Principle on Main-Sequence Stars and White Dwarfs

    Directory of Open Access Journals (Sweden)

    Mohamed Moussa

    2015-01-01

    Full Text Available This paper addresses the effect of generalized uncertainty principle, emerged from different approaches of quantum gravity within Planck scale, on thermodynamic properties of photon, nonrelativistic ideal gases, and degenerate fermions. A modification in pressure, particle number, and energy density are calculated. Astrophysical objects such as main-sequence stars and white dwarfs are examined and discussed as an application. A modification in Lane-Emden equation due to a change in a polytropic relation caused by the presence of quantum gravity is investigated. The applicable range of quantum gravity parameters is estimated. The bounds in the perturbed parameters are relatively large but they may be considered reasonable values in the astrophysical regime.

  4. Coseismic effects of the 2016 Amatrice seismic sequence: first geological results

    Directory of Open Access Journals (Sweden)

    EMERGEO W.G. :

    2016-11-01

    Full Text Available Since the beginning of the ongoing Amatrice seismic sequence on August 24, 2016, initiated by a Mw 6.0 normal faulting earthquake, the EMERGEO Working Group (an INGV team devoted to earthquake aftermath geological survey set off to investigate any coseismic effects on the natural environment. Up to now, we surveyed about 750 km2 and collected more than 3200 geological observations as differently oriented tectonic fractures together with intermediate- to small- sized landslides, that were mapped in the whole area. The most impressive coseismic evidence was found along the known active Mt. Vettore fault system, where surface ruptures with clear vertical/horizontal offset were observed for more than 5 km, while unclear and discontinuous coseismic features were recorded along the Laga Mts. Fault systems.

  5. Effects of bandwidth feedback on the automatization of an arm movement sequence.

    Science.gov (United States)

    Agethen, Manfred; Krause, Daniel

    2016-02-01

    We examined the effects of a bandwidth feedback manipulation on motor learning. Effects on movement accuracy, as well as on movement consistency, have been addressed in earlier studies. We have additionally investigated the effects on motor automatization. Because providing error feedback is believed to induce attentional control processes, we suppose that a bandwidth method should facilitate motor automatization. Participants (N=48) were assigned to four groups: one control group and three intervention groups. Participants of the intervention groups practiced an arm movement sequence with 760 trials. The BW0-Group practiced with 100% frequency of feedback. For the BW10-Group, feedback was provided when the errors were larger than 10°. The YokedBW10-Group participants were matched to the feedback schedule of research twins from the BW10-Group. All groups performed pre-tests and retention tests with a secondary task paradigm to test for automaticity. The BW10-Group indicated a higher degree of automatization compared with the BW0-Group, which did not exhibit a change in automaticity. The comparison of the YokedBW10-Group, which also exhibited automatization, and the BW10-Group leads to the proposal that reduction of quantitative feedback frequency and additional positive feedback are responsible for the bandwidth effect. Differences in movement accuracy and consistency were not evident.

  6. Effect of metal ions and petrochemicals on bioremediation of chlorpyrifos in aerobic sequencing batch bioreactor (ASBR).

    Science.gov (United States)

    Khalid, Saira; Hashmi, Imran; Jamal Khan, Sher; Qazi, Ishtiaq A; Nasir, Habib

    2016-10-01

    Application of chlorpyrifos (CP) has increased its environmental concentration. Increasing CP concentration has increased chances of adverse health effects. Its removal from environment has attained researcher's attention. CP degrading bacterial strains were isolated from wastewater and agricultural soil. Finally, selected five bacterial strains were identified using 16S rRNA nucleotide sequence analysis as Pseudomonas kilonensis SRK1, Serratia marcescens SRK2, Bacillus pumilus SRK4, Achromobacter xylosoxidans SRK5, and Klebsiella sp. T13. Interaction studies among bacterial strains demonstrated possibility for development of five membered bacterial consortium. Biodegradation potential of bacterial consortium was investigated in the presence of petrochemicals and trace metals. About 98 % CP removal was observed in sequencing batch reactors at inoculum level, 10 %; pH, 7; CP concentration, 400 mgL(-1), and HRT, 48 h. Experimental data has shown an excellent fit to first order growth model. Among all petrochemicals only toluene (in low concentration) has stimulatory effect on biodegradation of CP. Addition of petrochemicals (benzene, toluene, and xylene) in high concentration (100 mg L(-1)) inhibited bacterial activity and decreased CP removal. At low concentration i.e., 1 mg L(-1) of inorganic contaminants (Cu, Hg, and Zn) >96 % degradation was observed. Addition of Cu(II) in low concentration has stimulated CP removal efficiency. Hg(II) in all concentrations has strongly inhibited biodegradation rate except at 1 mgL(-1). In simulated pesticide, wastewater CP removal efficiency decreased to 77.5 %. Outcomes of study showed that both type and concentration of petrochemicals and trace metals influenced biodegradation of CP.

  7. Mood induction effects on motor sequence learning and stop signal reaction time.

    Science.gov (United States)

    Greeley, Brian; Seidler, Rachael D

    2017-01-01

    The neurobiological theory of positive affect proposes that positive mood states may benefit cognitive performance due to an increase of dopamine throughout the brain. However, the results of many positive affect studies are inconsistent; this may be due to individual differences. The relationship between dopamine and performance is not linear, but instead follows an inverted "U" shape. Given this, we hypothesized that individuals with high working memory capacity, a proxy measure for dopaminergic transmission, would not benefit from positive mood induction and in fact performance in dopamine-mediated tasks would decline. In contrast, we predicted that individuals with low working memory capacities would receive the most benefit after positive mood induction. Here, we explored the effect of positive affect on two dopamine-mediated tasks, an explicit serial reaction time sequence learning task and the stop signal task, predicting that an individual's performance is modulated not only by working memory capacity, but also on the type of mood. Improvements in explicit sequence learning from pre- to post-positive mood induction were associated with working memory capacity; performance declined in individuals with higher working memory capacities following positive mood induction, but improved in individuals with lower working memory capacities. This was not the case for negative or neutral mood induction. Moreover, there was no relationship between the change in stop signal reaction time with any of the mood inductions and individual differences in working memory capacity. These results provide partial support for the neurobiological theory of positive affect and highlight the importance of taking into account individual differences in working memory when examining the effects of positive mood induction.

  8. A model of human motor sequence learning explains facilitation and interference effects based on spike-timing dependent plasticity.

    Science.gov (United States)

    Wang, Quan; Rothkopf, Constantin A; Triesch, Jochen

    2017-08-01

    The ability to learn sequential behaviors is a fundamental property of our brains. Yet a long stream of studies including recent experiments investigating motor sequence learning in adult human subjects have produced a number of puzzling and seemingly contradictory results. In particular, when subjects have to learn multiple action sequences, learning is sometimes impaired by proactive and retroactive interference effects. In other situations, however, learning is accelerated as reflected in facilitation and transfer effects. At present it is unclear what the underlying neural mechanism are that give rise to these diverse findings. Here we show that a recently developed recurrent neural network model readily reproduces this diverse set of findings. The self-organizing recurrent neural network (SORN) model is a network of recurrently connected threshold units that combines a simplified form of spike-timing dependent plasticity (STDP) with homeostatic plasticity mechanisms ensuring network stability, namely intrinsic plasticity (IP) and synaptic normalization (SN). When trained on sequence learning tasks modeled after recent experiments we find that it reproduces the full range of interference, facilitation, and transfer effects. We show how these effects are rooted in the network's changing internal representation of the different sequences across learning and how they depend on an interaction of training schedule and task similarity. Furthermore, since learning in the model is based on fundamental neuronal plasticity mechanisms, the model reveals how these plasticity mechanisms are ultimately responsible for the network's sequence learning abilities. In particular, we find that all three plasticity mechanisms are essential for the network to learn effective internal models of the different training sequences. This ability to form effective internal models is also the basis for the observed interference and facilitation effects. This suggests that STDP, IP, and SN

  9. Solution and Solid-State Analysis of Binding of 13-Substituted Berberine Analogues to Human Telomeric G-quadruplexes.

    Science.gov (United States)

    Ferraroni, Marta; Bazzicalupi, Carla; Papi, Francesco; Fiorillo, Gaetano; Guamán-Ortiz, Luis Miguel; Nocentini, Alessio; Scovassi, Anna Ivana; Lombardi, Paolo; Gratteri, Paola

    2016-04-05

    The interaction between 13-phenylalkyl and 13-diphenylalkyl berberine derivatives (NAX) and human telomeric DNA G4 structures has been investigated by both spectroscopic and crystallographic methods. NAX042 and NAX053 are the best compounds improving the performance of the natural precursor berberine. This finding is in agreement with the X-ray diffraction result for the NAX053-Tel12 adduct, showing the ligand which interacts via π-stacking, sandwiched at the interface of two symmetry-related quadruplex units, with its benzhydryl group contributing to the overall stability of the adduct by means of additional π-stacking interactions with the DNA residues. The berberine derivatives were also investigated for their cytotoxic activity towards a panel of human cancer cell lines. Compounds NAX042 and NAX053 affect the viability of cancer cell lines in a dose-dependent manner.

  10. Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs

    DEFF Research Database (Denmark)

    Nguyen, Giang Huong; Tang, Weiliang; Robles, Ana I;

    2014-01-01

    Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathological...... features of Bloom syndrome, we analyzed mRNA and microRNA (miRNA) expression in fibroblasts from individuals with Bloom syndrome and in BLM-depleted control fibroblasts. We identified mRNA and miRNA expression differences in Bloom syndrome patient and BLM-depleted cells. Differentially expressed m...... dysfunction, and other features observed in Bloom syndrome individuals. BLM binds to G-quadruplex (G4) DNA, and G4 motifs were enriched at transcription start sites (TSS) and especially within first introns (false discovery rate ≤ 0.001) of differentially expressed mRNAs in Bloom syndrome compared with normal...

  11. A Study of Interaction between Flavonoids and the Parallel Quadruplex Structure [d(TGGGGT)]4 by Electrospray Ionization Mass Spectrometry

    Institute of Scientific and Technical Information of China (English)

    徐牛生; 杨洪梅; 崔勐; 宋凤瑞; 刘志强; 刘淑莹

    2012-01-01

    In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate interaction of 21 flavonoids (10 aglycones and 11 glycosides) with the parallel quadruplex structure [d(TGGGGT)]4. Relative binding affinities of flavonoids toward [d(TGGGGT)]4 were estimated based on the fraction of bound DNA. It was found that [d(TGGGGT)]4 showed a binding preference to the flavonoid glycosides over flavonoid aglycones. It was d duced that glycosylation played a key role for the [d(TGGGGT)]a-binding properties of flavonoid glycosides. Upon collision-induced dissociation, complexes of flavonoid/[d(TGGGGT)]4 underwent the loss of flavonoids, suggesting an end-stacking binding mode. The current work demonstrates that ESI-MS is a powerful tool in the study of irheraction between drugs and nucleic acids.

  12. Cell-SELEX-based selection and characterization of a G-quadruplex DNA aptamer against mouse dendritic cells.

    Science.gov (United States)

    Moghadam, M; Sankian, M; Abnous, K; Varasteh, A; Taghdisi, S M; Mahmoudi, M; Ramezani, M; Gholizadeh, Z; Ganji, A

    2016-07-01

    Targeting of dendritic cells (DCs) by aptamers increases antigen capture and presentation to the immune system. Our aim was to produce aptamers against DC molecules using the cell-SELEX procedure. For this purpose, 18 rounds of cell-SELEX were performed on mouse macrophage J774A.1 and CT26 as target and control cells, respectively. The selected aptamers were truncated and their binding to mouse macrophages, and immature and mature DCs analyzed. Two macrophage-specific aptamers, Seq6 and Seq7, were identified. A truncated form of Seq7, Seq7-4, 33 nucleotides in length and containing the G-quadruplex, bound macrophages and immature DCs with KD values in the nanomolar range. We anticipate that Seq7-4 has potential as a therapeutic tool in targeting of mouse macrophages and immature DCs to efficiently improve different immunotherapy approaches. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. State-of-the-art methodologies for the discovery and characterization of DNA G-quadruplex binders.

    Science.gov (United States)

    Pagano, Bruno; Cosconati, Sandro; Gabelica, Valerie; Petraccone, Luigi; De Tito, Stefano; Marinelli, Luciana; La Pietra, Valeria; di Leva, Francesco Saverio; Lauri, Ilaria; Trotta, Roberta; Novellino, Ettore; Giancola, Concetta; Randazzo, Antonio

    2012-01-01

    Nowadays, the molecular basis of interaction between low molecular weight compounds and biological macromolecules is the subject of numerous investigations aimed at the rational design of molecules with specific therapeutic applications. In the last decades, it has been demonstrated that DNA quadruplexes play a critical role in several biological processes both at telomeric and gene promoting levels thus providing a great stride in the discovery of ligands able to interact with such a biologically relevant DNA conformation. So far, a number of experimental and computational approaches have been successfully employed in order to identify new ligands and to characterize their binding to the DNA. The main focus of this review is the description of these methodologies, placing a particular emphasis on computational methods, isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopies.

  14. The 2010-2011 Canterbury Earthquake Sequence: Environmental effects, seismic triggering thresholds and geologic legacy

    Science.gov (United States)

    Quigley, Mark C.; Hughes, Matthew W.; Bradley, Brendon A.; van Ballegooy, Sjoerd; Reid, Catherine; Morgenroth, Justin; Horton, Travis; Duffy, Brendan; Pettinga, Jarg R.

    2016-03-01

    Seismic shaking and tectonic deformation during strong earthquakes can trigger widespread environmental effects. The severity and extent of a given effect relates to the characteristics of the causative earthquake and the intrinsic properties of the affected media. Documentation of earthquake environmental effects in well-instrumented, historical earthquakes can enable seismologic triggering thresholds to be estimated across a spectrum of geologic, topographic and hydrologic site conditions, and implemented into seismic hazard assessments, geotechnical engineering designs, palaeoseismic interpretations, and forecasts of the impacts of future earthquakes. The 2010-2011 Canterbury Earthquake Sequence (CES), including the moment magnitude (Mw) 7.1 Darfield earthquake and Mw 6.2, 6.0, 5.9, and 5.8 aftershocks, occurred on a suite of previously unidentified, primarily blind, active faults in the eastern South Island of New Zealand. The CES is one of Earth's best recorded historical earthquake sequences. The location of the CES proximal to and beneath a major urban centre enabled rapid and detailed collection of vast amounts of field, geospatial, geotechnical, hydrologic, biologic, and seismologic data, and allowed incremental and cumulative environmental responses to seismic forcing to be documented throughout a protracted earthquake sequence. The CES caused multiple instances of tectonic surface deformation (≥ 3 events), surface manifestations of liquefaction (≥ 11 events), lateral spreading (≥ 6 events), rockfall (≥ 6 events), cliff collapse (≥ 3 events), subsidence (≥ 4 events), and hydrological (10s of events) and biological shifts (≥ 3 events). The terrestrial area affected by strong shaking (e.g. peak ground acceleration (PGA) ≥ 0.1-0.3 g), and the maximum distances between earthquake rupture and environmental response (Rrup), both generally increased with increased earthquake Mw, but were also influenced by earthquake location and source

  15. Effects of Dosage Sequence on the Efficacy of Nonfumigant Nematicides, Plantain Yields, and Nematode Seasonal Fluctuations as influenced by Rainfall.

    Science.gov (United States)

    Badra, T; Caveness, F E

    1983-10-01

    Four nonfumigant nematicides applied three times during the wet season were used to study dosage sequence and nematicide effectiveness. Control of Helicotylenchus multicinctus (Cobb) Thorne and Meloidogyne javanica (Treub) Chitwood increased plantain (Musa AAB) yields. The nematicide (aldicarb, carbofuran, oxamyl, and miral) performance and yield response varied with dosage sequences. Applications of 2, 3, and 2 g ai/tree in March, July, and October (sequence I), respectively, gave greater control of M. javanica than did applications of 3, 2, and 2 g ai/tree in March, June, and September (sequence II), respectively. However, the high initial dose sequence was effective against H. multicinctus. Persistence of the different nematicides differed over the 14-month experimental period. Miral, aldicarb, and carbofuran were the most effective treatments against either species by the end of the wet and dry seasons. Dry season residual nematode populations were significantly lower in nematicide treated than in control plots. Yield increases over controls were 96.9, 90.1, 78.4, and 70.1% for carbofuran applied by sequence II, aldicarb by II and I, and oxantyl by II, respectively. Nematode populations directly fluctuated with rainfall and dropped to low (H. multicinctus) or to undetectable (M. javanica juveniles) levels during the dry season. Of the two nematodes studied, the more serious pest to plantain was H. multicinctus; it was tolerant to drought and survived the dry season in untreated soils.

  16. The Effects of Pitch Shifts on Delay-Induced Changes in Vocal Sequencing in a Songbird

    Science.gov (United States)

    Kelly, Conor W.

    2017-01-01

    Abstract Like human speech, vocal behavior in songbirds depends critically on auditory feedback. In both humans and songbirds, vocal skills are acquired by a process of imitation whereby current vocal production is compared to an acoustic target. Similarly, performance in adulthood relies strongly on auditory feedback, and online manipulations of auditory signals can dramatically alter acoustic production even after vocalizations have been well learned. Artificially delaying auditory feedback can disrupt both speech and birdsong, and internal delays in auditory feedback have been hypothesized as a cause of vocal dysfluency in persons who stutter. Furthermore, in both song and speech, online shifts of the pitch (fundamental frequency) of auditory feedback lead to compensatory changes in vocal pitch for small perturbations, but larger pitch shifts produce smaller changes in vocal output. Intriguingly, large pitch shifts can partially restore normal speech in some dysfluent speakers, suggesting that the effects of auditory feedback delays might be ameliorated by online pitch manipulations. Although birdsong provides a promising model system for understanding speech production, the interactions between sensory feedback delays and pitch shifts have not yet been assessed in songbirds. To investigate this, we asked whether the addition of a pitch shift modulates delay-induced changes in Bengalese finch song, hypothesizing that pitch shifts would reduce the effects of feedback delays. Compared with the effects of delays alone, combined delays and pitch shifts resulted in a significant reduction in behavioral changes in one type of sequencing (branch points) but not another (distribution of repeated syllables). PMID:28144622

  17. The Effects of Pitch Shifts on Delay-Induced Changes in Vocal Sequencing in a Songbird.

    Science.gov (United States)

    Wyatt, MacKenzie; Berthiaume, Emily A; Kelly, Conor W; Sober, Samuel J

    2017-01-01

    Like human speech, vocal behavior in songbirds depends critically on auditory feedback. In both humans and songbirds, vocal skills are acquired by a process of imitation whereby current vocal production is compared to an acoustic target. Similarly, performance in adulthood relies strongly on auditory feedback, and online manipulations of auditory signals can dramatically alter acoustic production even after vocalizations have been well learned. Artificially delaying auditory feedback can disrupt both speech and birdsong, and internal delays in auditory feedback have been hypothesized as a cause of vocal dysfluency in persons who stutter. Furthermore, in both song and speech, online shifts of the pitch (fundamental frequency) of auditory feedback lead to compensatory changes in vocal pitch for small perturbations, but larger pitch shifts produce smaller changes in vocal output. Intriguingly, large pitch shifts can partially restore normal speech in some dysfluent speakers, suggesting that the effects of auditory feedback delays might be ameliorated by online pitch manipulations. Although birdsong provides a promising model system for understanding speech production, the interactions between sensory feedback delays and pitch shifts have not yet been assessed in songbirds. To investigate this, we asked whether the addition of a pitch shift modulates delay-induced changes in Bengalese finch song, hypothesizing that pitch shifts would reduce the effects of feedback delays. Compared with the effects of delays alone, combined delays and pitch shifts resulted in a significant reduction in behavioral changes in one type of sequencing (branch points) but not another (distribution of repeated syllables).

  18. The congruency sequence effect 3.0: a critical test of conflict adaptation.

    Directory of Open Access Journals (Sweden)

    Wout Duthoo

    Full Text Available Over the last two decades, the congruency sequence effect (CSE -the finding of a reduced congruency effect following incongruent trials in conflict tasks- has played a central role in advancing research on cognitive control. According to the influential conflict-monitoring account, the CSE reflects adjustments in selective attention that enhance task focus when needed, often termed conflict adaptation. However, this dominant interpretation of the CSE has been called into question by several alternative accounts that stress the role of episodic memory processes: feature binding and (stimulus-response contingency learning. To evaluate the notion of conflict adaptation in accounting for the CSE, we construed versions of three widely used experimental paradigms (the colour-word Stroop, picture-word Stroop and flanker task that effectively control for feature binding and contingency learning. Results revealed that a CSE can emerge in all three tasks. This strongly suggests a contribution of attentional control to the CSE and highlights the potential of these unprecedentedly clean paradigms for further examining cognitive control.

  19. Electromyographic Patterns during Golf Swing: Activation Sequence Profiling and Prediction of Shot Effectiveness

    Directory of Open Access Journals (Sweden)

    Antanas Verikas

    2016-04-01

    Full Text Available This study analyzes muscle activity, recorded in an eight-channel electromyographic (EMG signal stream, during the golf swing using a 7-iron club and exploits information extracted from EMG dynamics to predict the success of the resulting shot. Muscles of the arm and shoulder on both the left and right sides, namely flexor carpi radialis, extensor digitorum communis, rhomboideus and trapezius, are considered for 15 golf players (∼5 shots each. The method using Gaussian filtering is outlined for EMG onset time estimation in each channel and activation sequence profiling. Shots of each player revealed a persistent pattern of muscle activation. Profiles were plotted and insights with respect to player effectiveness were provided. Inspection of EMG dynamics revealed a pair of highest peaks in each channel as the hallmark of golf swing, and a custom application of peak detection for automatic extraction of swing segment was introduced. Various EMG features, encompassing 22 feature sets, were constructed. Feature sets were used individually and also in decision-level fusion for the prediction of shot effectiveness. The prediction of the target attribute, such as club head speed or ball carry distance, was investigated using random forest as the learner in detection and regression tasks. Detection evaluates the personal effectiveness of a shot with respect to the player-specific average, whereas regression estimates the value of target attribute, using EMG features as predictors. Fusion after decision optimization provided the best results: the equal error rate in detection was 24.3% for the speed and 31.7% for the distance; the mean absolute percentage error in regression was 3.2% for the speed and 6.4% for the distance. Proposed EMG feature sets were found to be useful, especially when used in combination. Rankings of feature sets indicated statistics for muscle activity in both the left and right body sides, correlation-based analysis of EMG

  20. The Effect of the Explicit Instruction of Formulaic Sequences in Pre-Writing Vocabulary Activities on Foreign Language Writing

    Directory of Open Access Journals (Sweden)

    Dina Abdel Salam El-Dakhs

    2017-05-01

    Full Text Available The present study investigates the effect of the explicit instruction of formulaic sequences in pre-writing vocabulary activities on foreign language writing. To this end, a total of 81 Saudi pre-intermediate learners of English as a foreign language participated in a 10-week study of a pretest/posttest design. In every 2-hour session of a total of 10 sessions, the participants were required to read a news story and then re-write it individually without looking back at the original story. During the treatment period, the participants received different pre-writing vocabulary practice. One group, consisting of 44 students, practiced individual words in the news stories while the remaining 37 students studied formulaic sequences in the new stories before re-writing the stories in their own language. Analyzing the students’ writing showed that the explicit instruction of formulaic sequences led to an increased use of the sequences in students’ writing. The results also partially supported a positive influence for the explicit instruction of formulaic sequences on the learners’ lexical choices and overall writing quality. The practice provided on formulaic sequences in the study did not, however, result in any significant improvement in the learners’ use of formulaic sequences in autonomous story re-writing. Relevant pedagogical implications are proposed.

  1. Embedding strategies for effective use of information from multiple sequence alignments.

    OpenAIRE

    1997-01-01

    We describe a new strategy for utilizing multiple sequence alignment information to detect distant relationships in searches of sequence databases. A single sequence representing a protein family is enriched by replacing conserved regions with position-specific scoring matrices (PSSMs) or consensus residues derived from multiple alignments of family members. In comprehensive tests of these and other family representations, PSSM-embedded queries produced the best results overall when used with...

  2. The Effects of Common Knowledge Construction Model Sequence of Lessons on Science Achievement and Relational Conceptual Change

    Science.gov (United States)

    Ebenezer, Jazlin; Chacko, Sheela; Kaya, Osman Nafiz; Koya, Satya Kiran; Ebenezer, Devairakkam Luke

    2010-01-01

    The purpose of this study was to investigate the effects of the Common Knowledge Construction Model (CKCM) lesson sequence, an intervention based both in conceptual change theory and in Phenomenography, a subset of conceptual change theory. A mixed approach was used to investigate whether this model had a significant effect on 7th grade students'…

  3. The effect of vowel height on Voice Onset Time in stop consonants in CV sequences in spontaneous Danish

    DEFF Research Database (Denmark)

    Mortensen, Johannes; Tøndering, John

    2013-01-01

    Voice onset time has been reported to vary with the height of vowels following the stop consonant. This paper investigates the effects of vowel height on VOT in Danish CV sequences with stop consonants in Danish spontaneous speech. A significant effect of vowel height on VOT was found...

  4. The effect of disc inclination on the main sequence of star-forming galaxies

    Science.gov (United States)

    Morselli, L.; Renzini, A.; Popesso, P.; Erfanianfar, G.

    2016-11-01

    We use the Sloan Digital Sky Survey (York et al.) data base to explore the effect of the disc inclination angle on the derived star formation rate (SFR), hence on the slope and width of the main-sequence (MS) relation for star-forming galaxies. We find that SFRs for nearly edge-on discs are underestimated by factors ranging from ˜0.2 dex for low-mass galaxies up to ˜0.4 dex for high-mass galaxies. This results in a substantially flatter MS relation for high-inclination discs compared to that for less inclined ones, though the global effect over the whole sample of star-forming galaxies is relatively minor, given the small fraction of high-inclination discs. However, we also find that galaxies with high-inclination discs represent a non-negligible fraction of galaxies populating the so-called green valley, with derived SFRs intermediate between the MS and those of quenched, passively evolving galaxies.

  5. Treatment of opium alkaloid containing wastewater in sequencing batch reactor (SBR)-Effect of gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bural, Cavit B.; Demirer, Goksel N. [Middle East Technical University, Department of Environmental Engineering, 06531 Ankara (Turkey); Kantoglu, Omer [Turkish Atomic Energy Authority, Saraykoy Nuclear Research and Training Center, 06982, Kazan, Ankara (Turkey); Dilek, Filiz B., E-mail: fdilek@metu.edu.t [Middle East Technical University, Department of Environmental Engineering, 06531 Ankara (Turkey)

    2010-04-15

    Aerobic biological treatment of opium alkaloid containing wastewater as well as the effect of gamma irradiation as pre-treatment was investigated. Biodegradability of raw wastewater was assessed in aerobic batch reactors and was found highly biodegradable (83-90% degradation). The effect of irradiation (40 and 140 kGy) on biodegradability was also evaluated in terms of BOD{sub 5}/COD values and results revealed that irradiation imparted no further enhancement in the biodegradability. Despite the highly biodegradable nature of wastewater, further experiments in sequencing batch reactors (SBR) revealed that the treatment operation was not possible due to sludge settleability problem observed beyond an influent COD value of 2000 mg dm{sup -3}. Possible reasons for this problem were investigated, and the high molecular weight, large size and aromatic structure of the organic pollutants present in wastewater was thought to contribute to poor settleability. Initial efforts to solve this problem by modifying the operational conditions, such as SRT reduction, failed. However, further operational modifications including addition of phosphate buffer cured the settleability problem and influent COD was increased up to 5000 mg dm{sup -3}. Significant COD removal efficiencies (>70%) were obtained in both SBRs fed with original and irradiated wastewaters (by 40 kGy). However, pre-irradiated wastewater provided complete thebain removal and a better settling sludge, which was thought due to degradation of complex structure by radiation application. Degradation of the structure was observed by GC/MS analyses and enhancement in filterability tests.

  6. Implicit Structured Sequence Learning: An FMRI Study of the Structural Mere-Exposure Effect

    Directory of Open Access Journals (Sweden)

    Vasiliki eFolia

    2014-02-01

    Full Text Available In this event-related FMRI study we investigated the effect of five days of implicit acquisition on preference classification by means of an artificial grammar learning (AGL paradigm based on the structural mere-exposure effect and preference classification using a simple right-linear unification grammar. This allowed us to investigate implicit AGL in a proper learning design by including baseline measurements prior to grammar exposure. After 5 days of implicit acquisition, the FMRI results showed activations in a network of brain regions including the inferior frontal (centered on BA 44/45 and the medial prefrontal regions (centered on BA 8/32. Importantly, and central to this study, the inclusion of a naive preference FMRI baseline measurement allowed us to conclude that these FMRI findings were the intrinsic outcomes of the learning process itself and not a reflection of a preexisting functionality recruited during classification, independent of acquisition. Support for the implicit nature of the knowledge utilized during preference classification on day 5 come from the fact that the basal ganglia, associated with implicit procedural learning, were activated during classification, while the medial temporal lobe system, associated with explicit declarative memory, was consistently deactivated. Thus, preference classification in combination with structural mere-exposure can be used to investigate structural sequence processing (syntax in unsupervised AGL paradigms with proper learning designs.

  7. Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms.

    Science.gov (United States)

    Blice-Baum, Anna C; Mihailescu, Mihaela-Rita

    2014-01-01

    Fragile X syndrome, the most common form of inherited mental impairment in humans, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5'-untranslated region (UTR) and subsequent translational silencing of the fragile x mental retardation-1 (FMR1) gene. FMRP, which is proposed to be involved in the translational regulation of specific neuronal messenger RNA (mRNA) targets, contains an arginine-glycine-glycine (RGG) box RNA binding domain that has been shown to bind with high affinity to G-quadruplex forming mRNA structures. FMRP undergoes alternative splicing, and the binding of FMRP to a proposed G-quadruplex structure in the coding region of its mRNA (named FBS) has been proposed to affect the mRNA splicing events at exon 15. In this study, we used biophysical methods to directly demonstrate the folding of FMR1 FBS into a secondary structure that contains two specific G-quadruplexes and analyze its interactions with several FMRP isoforms. Our results show that minor splice isoforms, ISO2 and ISO3, created by the usage of the second and third acceptor sites at exon 15, bind with higher affinity to FBS than FMRP ISO1, which is created by the usage of the first acceptor site. FMRP ISO2 and ISO3 cannot undergo phosphorylation, an FMRP post-translational modification shown to modulate the protein translation regulation. Thus, their expression has to be tightly regulated, and this might be accomplished by a feedback mechanism involving the FMRP interactions with the G-quadruplex structures formed within FMR1 mRNA.

  8. A series of logic gates based on electrochemical reduction of Pb2+ in self-assembled G-quadruplex on the gold electrode.

    Science.gov (United States)

    Zhai, Wei; Du, Chunyan; Li, Xiaohong

    2014-02-28

    Direct reduction of Pb(2+) in self-assembled G-quadruplex on the gold electrode was first observed, which was applied in constructing a series of simple and reversible logic gates, such as one-input, two-input and three-input logic gates. Importantly, the largest scale of reversibility among two-input logic gates was achieved based on the reciprocal transformations of DNA.

  9. 金属配合物与G-四链体作用的研究进展%Interaction of metal complexes with G-quadruplex

    Institute of Scientific and Technical Information of China (English)

    孙静; 陈嘉曦; 陈伙炎; 毛宗万

    2012-01-01

    The activity of telomerase can be prohibited by the secondary structure of the telomere DNA-G-quadruplex formed by the single chain in G-rich field, resulting in the apoptosis in tumor cells. Compound which can induce the formation or stabilize the structure of G-quadruplex might be potential anticancer drug. The relative ease of synthesis, the variable structure and the positively charged center enable the metal complexes to in-teract with the grooves and loops of the quadruplex and negatively charged phosphates backbone. Therefore, many metal complexes have been designed and synthesized. This paper reviews the discoveries in the interaction of G-quadruplex with metal complexes in the past years.%端粒DNA形成的独特二级结构G-四链体对端粒酶的活性有明显抑制作用,能达到促进肿瘤细胞凋亡的作用.研究表明,能够诱使端粒DNA形成G-四链体及可以稳定这种结构的药物均可能成为有效的化疗药物.金属配合物由于其合成路径简单、结构多变、金属中心所携带的正电荷有利于与G-四链体的沟区、loop环以及带负电荷的磷酸骨架作用,增强其稳定性,因此越来越多的过渡金属配合物被设计用来研究与G-四链体作用.该文综述了近年来金属配合物与G-四链体DNA相互作用方面的研究进展.

  10. A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40)

    OpenAIRE

    Li, Xiang; Wang, Xiuxiu; Yang, Jielin; Liu, Yueming; He, Yuping; Pan, Liangwen

    2014-01-01

    Background To date, over 150 genetically modified (GM) crops are widely cultivated. To comply with regulations developed for genetically modified organisms (GMOs), including labeling policies, many detection methods for GMO identification and quantification have been developed. Results To detect the entrance and exit of unauthorized GM crop events in China, we developed a novel quadruplex real-time PCR method for simultaneous detection and quantification of GM cotton events GHB119 and T304-40...

  11. Boar seminal plasma exosomes: effect on sperm function and protein identification by sequencing.

    Science.gov (United States)

    Piehl, Lidia L; Fischman, M Laura; Hellman, Ulf; Cisale, Humberto; Miranda, Patricia V

    2013-04-15

    Mammalian seminal plasma contains membranous vesicles (exosomes), with a high content of cholesterol and sphingomyelin and a complex protein composition. Their physiological role is uncertain because sperm stabilization and activation effects have been reported. To analyze a putative modulatory role for semen exosomes on sperm activity in the boar, the effects of these vesicles on several sperm functional parameters were examined. Additionally, boar exosome proteins were sequenced and their incorporation into sperm was explored. Boar sperm were incubated under conditions that induce capacitation, manifested as increased tyrosine phosphorylation, cholesterol loss and greater fluidity in apical membranes, and the ability to undergo the lysophosphatidylcholine-induced acrosome reaction. After establishing this cluster of capacitation-dependent functional parameters, the effect produced by exosomes when present during or after sperm capacitation was analyzed. Exosomes inhibited the capacitation-dependent cholesterol efflux and fluidity increase in apical membranes, and the disappearance of a 14-kD phosphorylated polypeptide. In contrast, the acrosome reaction (spontaneous and lysophosphatidylcholine-induced) was not affected, and sperm binding to the oocyte zona pellucida was reduced only when vesicles were present during gamete coincubation. Liposomes with a lipid composition similar to that present in exosomes mimicked these effects, except the one on zona pellucida binding. Interaction between exosomes and sperm was confirmed by transfer of aminopeptidase activity. In addition, the major exosome protein, identified as actin, appeared to associate with sperm after coincubation. Exosome composition had a predominance for structural proteins (actin, plastin, ezrin, and condensin), enzymes, and several porcine seminal plasma-specific polypeptides (e.g., spermadhesins). Transfer of proteins from exosome to sperm and their ability to block cholesterol efflux supports a

  12. Fragile X mental retardation protein interactions with a G quadruplex structure in the 3'-untranslated region of NR2B mRNA.

    Science.gov (United States)

    Stefanovic, Snezana; DeMarco, Brett A; Underwood, Ayana; Williams, Kathryn R; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-12-01

    Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5'-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the post synaptic density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3'-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3'-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-d-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets.

  13. Label-free and ratiometric detection of nuclei acids based on graphene quantum dots utilizing cascade amplification by nicking endonuclease and catalytic G-quadruplex DNAzyme.

    Science.gov (United States)

    Wang, Guang-Li; Fang, Xin; Wu, Xiu-Ming; Hu, Xue-Lian; Li, Zai-Jun

    2016-07-15

    Herein, we report a ratiometric fluorescence assay based on graphene quantum dots (GQDs) for the ultrasensitive DNA detection by coupling the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for cascade signal amplifications. With o-phenylenediamine acted as the substrate of G-quadruplex/hemin DNAzyme, whose oxidization product (that is, 2,3-diaminophenazine, DAP) quenched the fluorescence intensity of GQDs (at 460nm) obviously, accompanied with the emergence of a new emission of DAP (at 564nm). The ratiometric signal variations at the emission wavelengths of 564 and 460nm (I564/I460) were utilized for label-free, sensitive, and selective detection of target DNA. Utilizing the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for amplified cascade generation of DAP, the proposed bioassay exhibited high sensitivity toward target DNA with a detection limit of 30fM. The method also had additional advantages such as facile preparation and easy operation.

  14. G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.

    Directory of Open Access Journals (Sweden)

    Aaron J Stevens

    Full Text Available We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5' end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

  15. G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter

    Science.gov (United States)

    Cree, Simone L.; Gibb, Andrew; Miller, Allison L.; Doudney, Kit; Aitchison, Alan; Eccles, Michael R.; Joyce, Peter R.; Filichev, Vyacheslav V.; Kennedy, Martin A.

    2014-01-01

    We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings. PMID:25437198

  16. [Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. II. the putative role of G-quadruplex structures in genomic rearrangements].

    Science.gov (United States)

    Varizhuk, A M; Sekridova, A V; Tankevich, M V; Podgorsky, V S; Smirnov, I P; Pozmogova, G E

    2016-11-01

    Three evolutionary conserved sites of Alu repeats (PQS2, PQS3 and PQS4) were shown to form stable inter- and intramolecular G-quadruplexes (GQs) in vitro. Structures and topologies of these GQs were elucidated using spectral methods. Self-association of G-rich Alu fragments was studied. Dimeric GQ formation from two distal identical or different putative quadruplex sites - (PQS2)2, (PQS3)2 or PQS2-PQS3 - within one lengthy DNA strand was demonstrated by a FRET-based method. Oligomer PQS4 (folded into a parallel intramolecular GQ) was shown to form stacks of quadruplexes that are stabilized by stacking interactions of external G-tetrads (this was confirmed by DOSY NMR, AFM microscopy and differential CD spectroscopy). Comparative analysis of the properties of various GQs allowed us to put forward a hypothesis of two general mechanisms of intermolecular GQ-dependant genomic rearrangements: 1) formation of a dimeric GQs; 2) association of pre-folded intramolecular parallel GQs from different strands into GQ-stacks. Thus, the observed co-localization of G-rich motifs of Alu elements with double-strand break hotspots and rearrangement hotspots may be accounted for by the specific secondary structure of these motifs. At the same time, this is likely primarily due to high abundance of such G-rich Alu fragments in the genome.

  17. Effects of Early and Late Rest Intervals on Performance and Overnight Consolidation of a Keyboard Sequence

    Science.gov (United States)

    Cash, Carla Davis

    2009-01-01

    Thirty-six nonmusicians practiced a five-element key-press sequence on a digital piano, repeating the sequence as quickly and accurately as possible during twelve 30-s practice blocks alternating with 30-s pauses. Twelve learners rested for 5 min between Blocks 3 and 4, another 12 learners rested for 5 min between Blocks 9 and 10, and the…

  18. Effects of Early and Late Rest Intervals on Performance and Overnight Consolidation of a Keyboard Sequence

    Science.gov (United States)

    Cash, Carla Davis

    2009-01-01

    Thirty-six nonmusicians practiced a five-element key-press sequence on a digital piano, repeating the sequence as quickly and accurately as possible during twelve 30-s practice blocks alternating with 30-s pauses. Twelve learners rested for 5 min between Blocks 3 and 4, another 12 learners rested for 5 min between Blocks 9 and 10, and the…

  19. Effects of remediation train sequence on decontamination of heavy metal-contaminated soil containing mercury.

    Science.gov (United States)

    Hseu, Zeng-Yei; Huang, Yu-Tuan; Hsi, Hsing-Cheng

    2014-09-01

    When a contaminated site contains pollutants including both nonvolatile metals and Hg, one single remediation technology may not satisfactorily remove all contaminants. Therefore, in this study, chemical extraction and thermal treatment were combined as a remediation train to remove heavy metals, including Hg, from contaminated soil. A 0.2 M solution of ethylenediamine tetraacetic acid (EDTA) was shown to be the most effective reagent for extraction of considerable amounts of Cu, Pb, and Zn (> 50%). Hg removal was ineffective using 0.2 M EDTA, but thermogravimetric analysis suggested that heating to 550 degrees C with a heating rate of 5 degrees C/min for a duration of 1 hr appeared to be an effective approach for Hg removal. With the employment of thermal treatment, up to 99% of Hg could be removed. However executing thermal treatment prior to chemical extraction reduced the effectiveness of the subsequent EDTA extraction because nonvolatile heavy metals were immobilized in soil aggregates after the 550 degrees C treatment. The remediation train of chemical extraction followed by thermal treatment appears to remediate soils that have been contaminated by many nonvolatile heavy metals and Hg. Implications: A remediation train conjoining two or more techniques has been initialized to remove multiple metals. Better understandings of the impacts of treatment sequences, namely, which technique should be employed first on the soil properties and the decontamination efficiency, are in high demand. This study provides a strategy to remove multiple heavy metals including Hg from a contaminated soil. The interactions between thermal treatment and chemical extraction on repartitioning of heavy metals was revealed. The obtained results could offer an integrating strategy to remediate the soil contaminated with both heavy metals and volatile contaminants.

  20. Effect of long-term exposure to mobile phone radiation on alpha-Int1 gene sequence of Candida albicans.

    Science.gov (United States)

    Shahin-Jafari, Ariyo; Bayat, Mansour; Shahhosseiny, Mohammad Hassan; Tajik, Parviz; Roudbar-Mohammadi, Shahla

    2016-05-01

    Over the last decade, communication industries have witnessed a tremendous expansion, while, the biological effects of electromagnetic waves have not been fully elucidated. Current study aimed at evaluating the mutagenic effect of long-term exposure to 900-MHz radiation on alpha-Int1 gene sequences of Candida albicans. A standard 900 MHz radiation generator was used for radiation. 10 ml volumes from a stock suspension of C. albicans were transferred into 10 polystyrene tubes. Five tubes were exposed at 4 °C to a fixed magnitude of radiation with different time periods of 10, 70, 210, 350 and 490 h. The other 5 tubes were kept far enough from radiation. The samples underwent genomic DNA extraction. PCR amplification of alpha-Int1 gene sequence was done using one set of primers. PCR products were resolved using agarose gel electrophoresis and the nucleotide sequences were determined. All samples showed a clear electrophoretic band around 441 bp and further sequencing revealed the amplified DNA segments are related to alpha-Int1 gene of the yeast. No mutations in the gene were seen in radiation exposed samples. Long-term exposure of the yeast to mobile phone radiation under the above mentioned conditions had no mutagenic effect on alpha-Int1 gene sequence.

  1. Differential effects of high-temperature stress on nuclear topology and transcription of repetitive noncoding and coding rye sequences.

    Science.gov (United States)

    Tomás, D; Brazão, J; Viegas, W; Silva, M

    2013-01-01

    The plant stress response has been extensively characterized at the biochemical and physiological levels. However, knowledge concerning repetitive sequence genome fraction modulation during extreme temperature conditions is scarce. We studied high-temperature effects on subtelomeric repetitive sequences (pSc200) and 45S rDNA in rye seedlings submitted to 40°C during 4 h. Chromatin organization patterns were evaluated through fluorescent in situ hybridization and transcription levels were assessed using quantitative real-time PCR. Additionally, the nucleolar dynamics were evaluated through fibrillarin immunodetection in interphase nuclei. The results obtained clearly demonstrated that the pSc200 sequence organization is not affected by high-temperature stress (HTS) and proved for the first time that this noncoding subtelomeric sequence is stably transcribed. Conversely, it was demonstrated that HTS treatment induces marked rDNA chromatin decondensation along with nucleolar enlargement and a significant increase in ribosomal gene transcription. The role of noncoding and coding repetitive rye sequences in the plant stress response that are suggested by their clearly distinct behaviors is discussed. While the heterochromatic conformation of pSc200 sequences seems to be involved in the stabilization of the interphase chromatin architecture under stress conditions, the dynamic modulation of nucleolar and rDNA topology and transcription suggest their role in plant stress response pathways.

  2. Effect of dissolved oxygen concentration on nitrite accumulation in nitrifying sequencing batch reactor.

    Science.gov (United States)

    Sánchez, Omar; Bernet, Nicolas; Delgenès, Jean-Philippe

    2007-08-01

    A mathematical model based on Activated Sludge Model No. 3 (International Water Association, London) and laboratory-scale experiments were used to investigate ammonia conversion by nitrification in a sequencing batch reactor (SBR). The purpose of the study was to assess the effect of dissolved oxygen concentration on nitrite accumulation in the SBR. As the dissolved oxygen concentration in the SBR depends on the balance between oxygen consumption and oxygen transfer rates, ammonium conversion was measured for different air flowrate values to obtain different dissolved oxygen concentration profiles during the cycle. The ammonia concentration in the feeding medium was 500 mg ammonium as nitrogen (N-NH4(+))/L, and the maximum nitrite concentration achieved during a cycle was approximately 50 mg nitrite as nitrogen (N-NO2)/L. The air flow supplied to the reactor was identified as a suitable parameter to control nitrite accumulation in the SBR. This identification was carried out based on experimental results and simulation with a calibrated model. At a low value of the volumetric mass-transfer coefficient (kLa), the maximum nitrite concentration achieved during a cycle depends strongly on k(L)a, whereas, at a high value of k(L)a, the maximum nitrite concentration was practically independent of kL(a).

  3. Effects of idle time on biological phosphorus removal by sequencing batch reactors.

    Science.gov (United States)

    Gao, Dawen; Yin, Hang; Liu, Lin; Li, Xing; Liang, Hong

    2013-12-01

    Three identical sequencing batch reactors (SBRs) were operated to investigate the effects of various idle times on the biological phosphorus (P) removal. The idle times were set to 3 hr (R1), 10 hr (R2) and 17 hr (R3). The results showed that the idle time of a SBR had potential impact on biological phosphorus removal, especially when the influent phosphorus concentration increased. The phosphorus removal efficiencies of the R2 and R3 systems declined dramatically compared with the stable R1 system, and the P-release and P-uptake rates of the R3 system in particular decreased dramatically. The PCR-DGGE analysis showed that uncultured Pseudomonas sp. (GQ183242.1) and beta-Proteobacteria (AY823971) were the dominant phosphorus removal bacteria for the R1 and R2 systems, while uncultured gamma-Proteobacteria were the dominant phosphorus removal bacteria for the R3 system. Glycogen-accumulating organisms (GAOs), such as uncultured Sphingomonas sp. (AM889077), were found in the R2 and R3 systems. Overall, the R1 system was the most stable and exhibited the best phosphorus removal efficiency. It was found that although the idle time can be prolonged to allow the formation of intracellular polymers when the phosphorus concentration of the influent is low, systems with a long idle time can become unstable when the influent phosphorus concentration is increased.

  4. Effects of La3+, Ce3+ on nitrogen removal in sequencing batch reactor

    Institute of Scientific and Technical Information of China (English)

    Qing XIA; Rui LIANG; Yuxiang MAO; Yuning HONG; Lili DING; Hongqiang REN; Mingyu ZHAO

    2009-01-01

    Batch experiments were conducted to study the short-term biological effects of rare earth ions (La3+,Ce3+) and their mixture on the nitrogen removal in a sequencing batch reactor (SBR). The data showed that higher NH4+-N removal rate, total inorganic nitrogen removal efficiency, and denitrification efficiency were achieved at lower concentrations of rare earth elements (REEs) ( < 1 mg/L). In the first hour of the aeration stage of SBR, the presence of REEs increased the total inorganic nitrogen removal efficiency and NH4+-N removal effi-ciency by 15.7% and 10%-15%, respectively. When the concentrations of REEs were higher than 1 mg/L, the total inorganic nitrogen removal efficiency decreased, and nitrate was found to accumulate in the effluent. When the concentrations of REEs was up to 50.0 mg/L, the total inorganic nitrogen removal efficiency was less than 30% of the control efficiency with a high level of nitrate. Lower concentrations of REEs were found to accelerate the nitrogen conversion and removal in SBR.

  5. Effects of extracellular polymer substances on aerobic granulation in sequencing batch reactors

    Institute of Scientific and Technical Information of China (English)

    WANG Zhi-ping; LIU Li-li; YAO Jie; SUN Li-xin; CAI Wei-min

    2009-01-01

    The effects of extracellular polymeric substances (EPS) on aerobic granulation in sequencing batch reactors (SBR) were investigated by evaluating the EPS content, and the relationship between EPS composition and surface properties of glucose-fed aerobic granules. The results show that aerobic granular sludge contains more EPS than seed sludge, and it is about 47 mg/gMLSS. Corresponding to the changes of EPS, the surface charge of microorganisms in granules increases from -0. 732 to -0. 845 meq/gMLSS, whereas the hydrophobicry changes significantly from 48.46% to 73. 16%. It is obviously that changes of EPS in sludge alter the negative surface charge and hydrophobieity of microorganisms in granules, enhance the polymeric interaction and promote the aerobic granulation. Moreover, EPS can serve as carbon and energy reserves in granulation, thus the growth between the interior and exterior bacteria is balanced, and the integrality of granules is maintained.SEM observation of the granules exhibits that EPS in granules are ropy ; by mixing with bacteria, compact matrix structure can be formed. The distribution of EPS in granules profiles the importance of EPS storage. It can be concluded that EPS play a crucial role in aerobic granulation.

  6. Tongue movements in vowel-consonant-vowel (VCV) sequences: The effect of consonant length

    Science.gov (United States)

    Lofqvist, Anders

    2005-09-01

    This study examined the effect of consonant duration on the tongue movement from the first to the second vowel in VCV sequences, where the consonant is a short or long labial nasal consonant. Lip, tongue, and jaw movements were recorded in native speakers of Japanese using a magnetometer system. Measurements were made of the duration, path, and speed of the tongue movement trajectory between the two vowels. The coordination of the onsets of the lip closing and tongue movements was also studied, as well as the relative part of the trajectory that occurred during the consonant and the vowels. Preliminary results show a robust difference in duration between the long and short consonants, with the long ones about twice as long. The duration of the tongue movement was longer in the long than the short consonants. Both the peak and average speed of the tongue movement were slower in the long consonants. The tongue movement path was slightly longer in the long consonants. These results suggest that speakers adjust the tongue movement trajectory so that a similar relationship between the movement and the consonant closure is maintained in both the long and the short consonants. [Work supported by NIH.

  7. Sequencing, speech production, and selective effects of aging on phonological and morphological speech errors.

    Science.gov (United States)

    MacKay, Donald G; James, Lori E

    2004-03-01

    To test age-linked predictions of node structure theory (NST) and other theories, young and older adults performed a task that elicited large numbers of phonological and morphological speech errors. Stimuli were visually presented words containing either /p/ or /b/, and participants changed the /p/ to /b/ or vice versa and produced the resulting word as quickly as possible. For example, the correct response was "bunk" for the stimulus PUNK, and "ripped" for RIBBED. Consistent with NST predictions, the elicited speech errors exhibited selective effects of aging. Some error types decreased with aging. For example, young adults produced more nonsequential substitution errors (as a percentage of total errors) than older adults (e.g., intended bills misproduced as "gills"). However, other error types remained constant or increased with aging. For example, older adults produced more omission errors than young adults, especially omissions involving inflectional endings (e.g. intended ripped misproduced as "np"). In addition, older adults exhibited special difficulties with 2 types of phonological and morphological sequencing processes.

  8. Training Sequences and their Effects on Task Performance and User Outcomes

    DEFF Research Database (Denmark)

    Sanford, Clive Carlton

    2007-01-01

    This article introduces the concept of information technology (IT) training sequencesand examines how sequencing of conceptual and procedural training impact IT task performance, user satisfaction and users' self-efficacy. Using assimilation theory, we develop four hypotheses related to training...... sequences. These hypotheses were then tested in a database design context using a quasi-experimental study involving student subjects. Empirical results demonstrate improved far-transfer andnear-transfer task performance and higher self-efficacy for subjects trained in the conceptual-procedural sequence vs...

  9. G-quadruplex (G4) motifs in the maize (Zea mays L.) genome are enriched at specific locations in thousands of genes coupled to energy status, hypoxia, low sugar, and nutrient deprivation.

    Science.gov (United States)

    Andorf, Carson M; Kopylov, Mykhailo; Dobbs, Drena; Koch, Karen E; Stroupe, M Elizabeth; Lawrence, Carolyn J; Bass, Hank W

    2014-12-20

    The G-quadruplex (G4) elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationally identified 149,988 non-telomeric G4 motifs in maize (Zea mays L., B73 AGPv2), 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5' UTR and the other at the 5' end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia (group VII ERFs), oxidative stress (DJ-1/GATase1), and energy status (AMPK/SnRK) pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel.

  10. Effects of Cr and Ni on the efficiency and performance of sequencing ...

    African Journals Online (AJOL)

    Yomi

    2012-03-15

    Mar 15, 2012 ... Key words: Adsorption, chromium, heavy metals, nickel, sequencing batch reactor (SBR) system. INTRODUCTION ... Industrial estates often have good internal road ... resistance to the shock load and toxic substance (Metcalf.